Modulators of ATP-binding cassette transporters

ABSTRACT

The present invention relates to modulators of ATP-Binding Cassette (“ABC”) transporters or fragments thereof, including Cystic Fibrosis Transmembrane Conductance Regulator (“CFTR”), compositions thereof, and methods therewith. The present invention also relates to methods of treating ABC transporter mediated diseases using such modulators.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. § 119 of U.S.Provisional Application No. 60/540,564, filed Jan. 30, 2004, and U.S.Provisional Application No. 60/603,503, filed Aug. 20, 2004, the entirecontents of the above two applications being incorporated herein byreference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to modulators of ATP-Binding Cassette(“ABC”) transporters or fragments thereof, including Cystic FibrosisTransmembrane Conductance Regulator (“CFTR”), compositions thereof, andmethods therewith. The present invention also relates to methods oftreating ABC transporter mediated diseases using such modulators.

BACKGROUND OF THE INVENTION

ABC transporters are a family of membrane transporter proteins thatregulate the transport of a wide variety of pharmacological agents,potentially toxic drugs, and xenobiotics, as well as anions. ABCtransporters are homologous membrane proteins that bind and use cellularadenosine triphosphate (ATP) for their specific activities. Some ofthese transporters were discovered as multidrug resistance proteins(like the MDR1-P glycoprotein, or the multidrug resistance protein,MRP1), defending malignant cancer cells against chemotherapeutic agents.To date, 48 ABC Transporters have been identified and grouped into 7families based on their sequence identity and function.

ABC transporters regulate a variety of important physiological roleswithin the body and provide defense against harmful environmentalcompounds. Because of this, they represent important potential drugtargets for the treatment of diseases associated with defects in thetransporter, prevention of drug transport out of the target cell, andintervention in other diseases in which modulation of ABC transporteractivity may be beneficial.

One member of the ABC transporter family commonly associated withdisease is the cAMP/ATP-mediated anion channel, CFTR. CFTR is expressedin a variety of cells types, including absorptive and secretoryepithelia cells, where it regulates anion flux across the membrane, aswell as the activity of other ion channels and proteins. In epitheliacells, normal functioning of CFTR is critical for the maintenance ofelectrolyte transport throughout the body, including respiratory anddigestive tissue. CFTR is composed of approximately 1480 amino acidsthat encode a protein made up of a tandem repeate of transmembranedomains, each containing six transmembrane helices and a nucleotidebinding domain. The two transmembrane domains are linked by a large,polar, regulatory (R)-domain with multiple phosphorylation sites thatregulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in CysticFibrosis (“CF”), the most common fatal genetic disease in humans. CysticFibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia leads to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhanced mucus accumulationin the lung and the accompanying microbial infections that ultimatelycause death in CF patients. In addition to respiratory disease, CFpatients typically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, >1000 disease causingmutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/). The most prevalent mutation isa deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the endoplasmic reticulum (“ER”), and traffic to theplasma membrane. As a result, the number of channels present in themembrane is far less than observed in cells expressing wild-type CFTR.In addition to impaired trafficking, the mutation results in defectivechannel gating. Together, the reduced number of channels in the membraneand the defective gating lead to reduced anion transport acrossepithelia leading to defective ion and fluid transport. (Quinton, P. M.(1990), FASEB J. 4: 2709-2727). Studies have shown, however, that thereduced numbers of ΔF508-CFTR in the membrane are functional, albeitless than wild-type CFTR. (Dalemans et al. (1991), Nature Lond. 354:526-528; Denning et al., supra; Pasyk and Foskett (1995), J. Cell.Biochem. 270: 12347-50). In addition to ΔF508-CFTR, other diseasecausing mutations in CFTR that result in defective trafficking,synthesis, and/or channel gating could be up- or down-regulated to alteranion secretion and modify disease progression and/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl— channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

In addition to Cystic Fibrosis, modulation of CFTR activity may bebeneficial for other diseases not directly caused by mutations in CFTR,such as secretory diseases and other protein folding diseases mediatedby CFTR. These include, but are not limited to, chronic obstructivepulmonary disease (COPD), dry eye disease, and Sjögren's Syndrome.

COPD is characterized by airflow limitation that is progressive and notfully reversible. The airflow limitation is due to mucus hypersecretion,emphysema, and bronchiolitis. Activators of mutant or wild-type CFTRoffer a potential treatment of mucus hypersecretion and impairedmucociliary clearance that is common in COPD. Specifically, increasinganion secretion across CFTR may facilitate fluid transport into theairway surface liquid to hydrate the mucus and optimized periciliaryfluid viscosity. This would lead to enhanced mucociliary clearance and areduction in the symptoms associated with COPD. Dry eye disease ischaracterized by a decrease in tear aqueous production and abnormal tearfilm lipid, protein and mucin profiles. There are many causes of dryeye, some of which include age, Lasik eye surgery, arthritis,medications, chemical/thermal burns, allergies, and diseases, such ascystic fibrosis and Sjögrens's syndrome. Increasing anion secretion viaCFTR would enhance fluid transport from the corneal endothelial cellsand secretory glands surrounding the eye to increase corneal hydration.This would help to alleviate the symptoms associated with dry eyedisease. Sjögrens's syndrome is an autoimmune disease in which theimmune system attacks moisture-producing glands throughout the body,including the eye, mouth, skin, respiratory tissue, liver, vagina, andgut. Symptoms, include, dry eye, mouth, and vagina, as well as lungdisease. The disease is also associated with rheumatoid arthritis,systemic lupus, systemic sclerosis, and polymypositis/dermatomyositis.Defective protein trafficking is believed to cause the disease, forwhich treatment options are limited. Modulators of CFTR activity mayhydrate the various organs afflicted by the disease and help to elevatethe associated symptoms.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery, has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases. The two ways that the ER machinery can malfunction is eitherby loss of coupling to ER export of the proteins leading to degradation,or by the ER accumulation of these defective/misfolded proteins [AridorM, et al., Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et al.,Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J., et al.,Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et al., TIPS, 21,pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp. 186-198(1999)]. The diseases associated with the first class of ER malfunctionare cystic fibrosis (due to misfolded ΔF508-CFTR as discussed above),hereditary emphysema (due to a1-antitrypsin; non Piz variants),hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, suchas protein C deficiency, Type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, Type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses (due to lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are glycanosis CDGtype 1, hereditary emphysema (due to α1-antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-antichymotrypsin), diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A), Straussler-Scheinkersyndrome, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome.

In addition to up-regulation of CFTR activity, reducing anion secretionby CFTR modulators may be beneficial for the treatment of secretorydiarrheas, in which epithelial water transport is dramatically increasedas a result of secretagogue activated chloride transport. The mechanisminvolves elevation of cAMP and stimulation of CFTR.

Although there are numerous causes of diarrhea, the major consequencesof diarrheal diseases, resulting from excessive chloride transport arecommon to all, and include dehydration, acidosis, impaired growth anddeath.

Acute and chronic diarrheas represent a major medical problem in manyareas of the world. Diarrhea is both a significant factor inmalnutrition and the leading cause of death (5,000,000 deaths/year) inchildren less than five years old.

Secretory diarrheas are also a dangerous condition in patients ofacquired immunodeficiency syndrome (AIDS) and chronic inflammatory boweldisease (IBD). Sixteen million travelers to developing countries fromindustrialized nations every year develop diarrhea, with the severityand number of cases of diarrhea varying depending on the country andarea of travel.

Diarrhea in barn animals and pets such as cows, pigs and horses, sheep,goats, cats and dogs, also known as scours, is a major cause of death inthese animals. Diarrhea can result from any major transition, such asweaning or physical movement, as well as in response to a variety ofbacterial or viral infections and generally occurs within the first fewhours of the animal's life.

The most common diarrheal causing bacteria is enterotoxogenic E-coli(ETEC) having the K99 pilus antigen. Common viral causes of diarrheainclude rotavirus and coronavirus. Other infectious agents includecryptosporidium, giardia lamblia, and salmonella, among others.

Symptoms of rotaviral infection include excretion of watery feces,dehydration and weakness. Coronavirus causes a more severe illness inthe newborn animals, and has a higher mortality rate than rotaviralinfection. Often, however, a young animal may be infected with more thanone virus or with a combination of viral and bacterial microorganisms atone time. This dramatically increases the severity of the disease.

Accordingly, there is a need for modulators of an ABC transporteractivity, and compositions thereof, that can be used to modulate theactivity of the ABC transporter in the cell membrane of a mammal.

There is a need for methods of treating ABC transporter mediateddiseases using such modulators of ABC transporter activity.

There is a need for methods of modulating an ABC transporter activity inan ex vivo cell membrane of a mammal.

There is a need for modulators of CFTR activity that can be used tomodulate the activity of CFTR in the cell membrane of a mammal.

There is a need for methods of treating CFTR-mediated diseases usingsuch modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivocell membrane of a mammal.

DESCRIPTION OF FIGURES

FIG. 1 recites compounds excluded from certain embodiments of thepresent invention.

FIG. 2 recites exemplary compounds of the present invention, along withselected analytical data and activity data.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are useful asmodulators of ABC transporter activity. These compounds have the generalformula I:

or a pharmaceutically acceptable salt thereof, wherein Ht, R^(N), ringA, ring B, X, R^(X), and x are described below.

These compounds and pharmaceutically acceptable compositions are usefulfor treating or lessening the severity of a variety of diseases,disorders, or conditions, including, but not limited to, cysticfibrosis, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, diabetesmellitus, laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, hereditaryemphysema, congenital hyperthyroidism, osteogenesis imperfecta,hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus (di),neurophyseal di, neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's-disease, Parkinson's disease, amyotrophic lateral sclerosis,progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders asuch as Huntington, spinocerebullar ataxia typeI, spinal and bulbar muscular atrophy, dentatorubal pallidoluysian, andmyotonic dystrophy, as well as spongiform encephalopathies, such ashereditary Creutzfeldt-Jakob disease, Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, and Sjogren'sdisease.

DESCRIPTION OF THE DRAWINGS

FIG. 1 recites the compounds excluded from certain embodiments of thepresent invention, as described below.

DETAILED DESCRIPTION OF THE INVENTION 1. General Description ofCompounds of the Invention

The present invention relates to compounds of formula I useful asmodulators of ABC transporter activity:

or a pharmaceutically acceptable salt thereof, wherein:

Ht is a 5-membered heteroaro matic ring containing 1-4 heteroatomsselected from O, S, N, or NR, wherein said ring is optionally fused to a6-membered monocyclic or 10-membered bicyclic, carbocyclic orheterocyclic, aromatic or non-aromatic ring, wherein Ht is optionallysubstituted with w occurrences of —WR^(W), wherein w is 0-5;

R^(N) is H or R;

R is hydrogen or C₁₋₆ aliphatic wherein up to two methylene units of Rare optionally and independently replaced by —CO—, —CS—, —COCO—,—CONR′—, —CONR′NR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—,—NR′NR′, —NR′NR′CO—, —NR′CO—, —S—, —SO, —SO₂—, —NR′—, —SO₂NR′—, NR′SO₂—,or —NR′SO₂NR′—;

ring A is 3-7 membered monocyclic ring having 0-3 heteroatoms selectedfrom O, S, N, or NH, wherein ring A is optionally substituted with qoccurrences of -QR^(Q);

ring B is optionally fused to 5-6 membered carbocyclic or heterocyclic,aromatic or non-aromatic ring;

each of x, q, and w is independently 0-5;

each —X—R^(X), -Q-R^(Q), and —W—R^(W) is independently R′;

R′ is independently R¹, R², R³, R⁴, or R⁵;

R¹ is oxo, R⁶ or ((C1-C4)aliphatic)_(n)-Y;

n is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶, NH₂, NHR⁶,N(R⁶)₂, NR⁶R⁸, COOH, COOR⁶ or OR⁶; or

two R¹ on adjacent ring atoms, taken together, form 1,2-methylenedioxyor 1,2-ethylenedioxy;

R² is aliphatic, wherein each R² optionally comprises up to 2substituents independently selected from R¹, R⁴, or R⁵;

R³ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ringoptionally comprising up to 3 substituents, independently selected fromR¹, R², R⁴ or R⁵;

R⁴ is OR⁵, OR⁶, OC(O)R⁶, OC(O)R⁵, OC(O)OR⁶, OC(O)OR⁵, OC(O)N(R⁶)₂,OC(O)N(R⁵)₂, OC(O)N(R⁶R⁵), SR⁶, SR⁵, S(O)R⁶, S(O)R⁵, SO₂R⁶, SO₂R⁵,SO₂N(R⁶)₂, SO₂N(R⁵)₂, SO₂NR⁵R⁶, SO₃R⁶, SO₃R⁵, C(O)R⁵, C(O)OR⁵, C(O)R⁶,C(O)OR⁶, C(O)N(R⁶)₂, C(O)N(R⁵)₂, C(O)N(R⁵R⁶), C(O)N(OR⁶)R⁶,C(O)N(OR⁵)R⁶, C(O)N(OR⁶)R⁵, C(O)N(OR⁵)R⁵, C(NOR⁶)R⁶, C(NOR⁶)R⁵,C(NOR⁵)R⁶, C(NOR⁵)R⁵, N(R⁶)₂, N(R⁵)₂, N(R⁵R⁶), NR⁵C(O)R⁵, NR⁶C(O)R⁶,NR⁶C(O)R⁵, NR⁵C(O)R⁶, NR⁶C(O)OR⁶, NR⁵C(O)OR⁶, NR⁶C(O)OR⁵, NR⁵C(O)OR⁵,NR⁶C(O)N(R⁶)₂, NR⁶C(O)NR⁵R⁶, NR⁶C(O)N(R⁵)₂, NR⁵C(O)N(R⁶)₂, NR⁵C(O)NR⁵R⁶,NR⁵C(O)N(R⁵)₂, NR⁶SO₂R⁶, NR⁶SO₂R⁵, NR⁵SO₂R⁶,NR⁵SO₂R⁵, NR⁶SO₂N(R⁶)₂,NR⁶SO₂NR⁵R⁶, NR⁶SO₂N(R⁵)₂, NR⁵SO₂N(R⁶)₂, NR⁵SO₂NR⁵R⁶, NR⁵SO₂N(R⁵)₂,N(OR⁶)R⁶, N(OR⁶)R⁵, N(OR⁵)R⁵, or N(OR⁵)R⁶;

R⁵ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring,optionally comprising up to 3 R¹ substituents;

R⁶ is H or aliphatic, wherein R⁶ optionally comprises a R⁷ substituent;

R⁷ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and eachR⁷ optionally comprises up to 2 substituents independently chosen fromH, (C1-C6)-straight or branched alkyl, (C₂-₆) straight or branchedalkenyl or alkynyl, 1,2-methylenedioxy, 1,2-ethylenedioxy, or(CH₂)_(n)-Z;

Z is selected from halo, CN, NO₂, CF₃, OCF₃, OH, S-aliphatic,S(O)-aliphatic, SO₂-aliphatic, NH₂, NH-aliphatic, N(aliphatic)₂,N(aliphatic)R⁸, NHR⁸, COOH, C(O)O(-aliphatic), or O-aliphatic; and

R⁸ is an amino protecting group.

In another embodiment, the present invention provides compounds offormula I, wherein the compounds set forth in FIG. 1 are excluded.

2. Compounds and Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated.

The term “ABC-transporter” as used herein means an ABC-transporterprotein or a fragment thereof comprising at least one binding domain,wherein said protein or fragment thereof is present in vivo or in vitro.The term “binding domain” as used herein means a domain on theABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. etal., J. Gen. Physiol. (1998): 111(3), 477-90.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see,e.g., http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

The term “modulating” as used herein means increasing or decreasing by ameasurable amount.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausalito: 1999, and “March'sAdvanced Organic Chemistry”, 5^(th) Ed., Ed.: Smith, M. B. and March,J., John Wiley & Sons, New York: 2001, the entire contents of which arehereby incorporated by reference.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and preferablytheir recovery, purification, and use for one or more of the purposesdisclosed herein. In some embodiments, a stable compound or chemicallyfeasible compound is one that is not substantially altered when kept ata temperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-20 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-10aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-8 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-6 aliphatic carbon atoms, and in yet other embodimentsaliphatic groups contain 1-4 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule wherein any individual ring in said bicyclic ringsystem has 3-7 members. Suitable aliphatic groups include, but are notlimited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “heteroaliphatic”, as used herein, means aliphatic groupswherein one or two carbon atoms are independently replaced by one ormore of oxygen, sulfur, nitrogen, phosphorus, or silicon.Heteroaliphatic groups may be substituted or unsubstituted, branched orunbranched, cyclic or acyclic, and include “heterocycle”,“heterocyclyl”, “heterocycloaliphatic”, or “heterocyclic” groups.

The term “heterocycle”, “heterocyclyl”, “heterocycloaliphatic”, or“heterocyclic” as used herein means non-aromatic, monocyclic, bicyclic,or tricyclic ring systems in which one or more ring members is anindependently selected heteroatom. In some embodiments, the“heterocycle”, “heterocyclyl”, “heterocycloaliphatic”, or “heterocyclic”group has three to fourteen ring members in which one or more ringmembers is a heteroatom independently selected from oxygen, sulfur,nitrogen, or phosphorus, and each ring in the system contains 3 to 7ring members.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

The term “alkoxy”, or “thioalkyl”, as used herein, refers to an alkylgroup, as previously defined, attached to the principal carbon chainthrough an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.

The terms “haloaliphatic” and “haloalkoxy” means aliphatic or alkoxy, asthe case may be, substituted with one or more halogen atoms. The term“halogen” means F, Cl, Br, or I. Examples of haloaliphatic incude —CHF₂,—CH₂F, —CF₃, —CF₂—, or perhaloalkyl, such as, —CF₂CF₃.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains 3 to 7 ring members. The term“aryl” may be used interchangeably with the term “aryl ring”. The term“aryl” also refers to heteroaryl ring systems as defined hereinbelow.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms, and whereineach ring in the system contains 3 to 7 ring members. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic”.

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) orheteroaryl (including heteroaralkyl and heteroarylalkoxy and the like)group may contain one or more substituents. Suitable substituents on theunsaturated carbon atom of an aryl or heteroaryl group are selected fromhalogen; —R^(o); —OR^(o); —SR^(o); 1,2-methylene-dioxy;1,2-ethylenedioxy; phenyl (Ph) optionally substituted with R^(o); —O(Ph)optionally substituted with R^(o); —(CH₂)₁₋₂(Ph), optionally substitutedwith R^(o); —CH═CH(Ph), optionally substituted with R^(o); —NO₂; —CN;—N(R^(o))₂; —NR^(o)C(O)R^(o); —NR^(o)C(O)N(R^(o))₂; —NR^(o)CO₂R^(o);—NR^(o)NR^(o)C(O)R^(o); —NR^(o)NR^(o)C(O)N(R^(o))₂;—NR^(o)NR^(o)CO₂R^(o); —C(O)C(O)R^(o); —C(O)CH₂C(O)R^(o); —CO₂R^(o);—C(O)R^(o); —C(O)N(R^(o))₂; —OC(O)N(R^(o))₂; —S(O)₂R^(o); —SO₂N(R^(o))₂;—S(O)R^(o); —NR^(o)SO₂N(R^(o))₂; —NR^(o)SO₂R^(o); —C(═S)N(R^(o))₂;—C(═NH)—N(R^(o))₂; or —(CH₂)₀₋₂NHC(O)R^(o) wherein each independentoccurrence of R^(o) is selected from hydrogen, optionally substitutedC₁₋₆ aliphatic, an unsubstituted 5-6 membered heteroaryl or heterocyclicring, phenyl, —O(Ph), or —CH₂(Ph), or, notwithstanding the definitionabove, two independent occurrences of R^(o), on the same substituent ordifferent substituents, taken together with the atom(s) to which eachR^(o) group is bound, form a 3-8-membered cycloalkyl, heterocyclyl,aryl, or heteroaryl ring having 0-3 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Optional substituents on the aliphaticgroup of R^(o) are selected from NH₂, NH(C₁₋₄aliphatic),N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH, O(C₁₋₄aliphatic), NO₂,CN, CO₂H, CO₂(C₁₋₄aliphatic), O(haloC₁₋₄ aliphatic), orhaloC₁₋₄aliphatic, wherein each of the foregoing C₁₋₄aliphatic groups ofR^(o) is unsubstituted.

An aliphatic or heteroaliphatic group, or a non-aromatic heterocyclicring may contain one or more substituents. Suitable substituents on thesaturated carbon of an aliphatic or heteroaliphatic group, or of anon-aromatic heterocyclic ring are selected from those listed above forthe unsaturated carbon of an aryl or heteroaryl group and additionallyinclude the following: ═O, ═S, ═NNHR*, ═NN(R*)₂, ═NNHC(O)R*,═NNHCO₂(alkyl), ═NNHSO₂(alkyl), or ═NR*, where each R* is independentlyselected from hydrogen or an optionally substituted C₁₋₆ aliphatic.Optional substituents on the aliphatic group of R* are selected fromNH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halogen, C₁₋₄ aliphatic,OH, O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄ aliphatic), O(halo C₁₋₄aliphatic), or halo(C₁₋₄ aliphatic), wherein each of the foregoingC₁₋₄aliphatic groups of R* is unsubstituted.

Optional substituents on the nitrogen of a non-aromatic heterocyclicring are selected from —R⁺, —N(R⁺)₂, —C(O)R⁺, —CO₂R⁺, —C(O)C(O)R⁺,—C(O)CH₂C(O)R⁺, —SO₂R⁺, —SO₂N(R⁺)₂, —C(═S)N(R⁺)₂, —C(═NH)—N(R⁺)₂, or—NR⁺SO₂R⁺; wherein R⁺ is hydrogen, an optionally substituted C₁₋₆aliphatic, optionally substituted phenyl, optionally substituted —O(Ph),optionally substituted —CH₂(Ph), optionally substituted —(CH₂)₁₋₂(Ph);optionally substituted —CH═CH(Ph); or an unsubstituted 5-6 memberedheteroaryl or heterocyclic ring having one to four heteroatomsindependently selected from oxygen, nitrogen, or sulfur, or,notwithstanding the definition above, two independent occurrences of R⁺,on the same substituent or different substituents, taken together withthe atom(s) to which each R⁺ group is bound, form a 3-8-memberedcycloalkyl, heterocyclyl, aryl, or heteroaryl ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur.Optional substituents on the aliphatic group or the phenyl ring of R⁺are selected from NH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halogen,C₁₋₄ aliphatic, OH, O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄aliphatic), O(halo C₁₋₄ aliphatic), or halo(C₁₋₄ aliphatic), whereineach of the foregoing C₁₋₄aliphatic groups of R⁺ is unsubstituted.

The term “alkylidene chain” refers to a straight or branched carbonchain that may be fully saturated or have one or more units ofunsaturation and has two points of attachment to the rest of themolecule. The term “spirocycloalkylidene” refers to a carbocyclic ringthat may be fully saturated or have one or more units of unsaturationand has two points of attachment from the same ring carbon atom to therest of the molecule.

As detailed above, in some embodiments, two independent occurrences ofR^(o) (or R⁺, or any other variable similarly defined herein), are takentogether together with the atom(s) to which each variable is bound toform a 3-8-membered cycloalkyl, heterocyclyl, aryl, or heteroaryl ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur. Exemplary rings that are formed when two independent occurrencesof R^(o) (or R⁺, or any other variable similarly defined herein) aretaken together with the atom(s) to which each variable is bound include,but are not limited to the following: a) two independent occurrences ofR^(o) (or R⁺, or any other variable similarly defined herein) that arebound to the same atom and are taken together with that atom to form aring, for example, N(R^(o))₂, where both occurrences of R^(o) are takentogether with the nitrogen atom to form a piperidin-1-yl,piperazin-1-yl, or morpholin-4-yl group; and b) two independentoccurrences of R^(o) (or R⁺, or any other variable similarly definedherein) that are bound to different atoms and are taken together withboth of those atoms to form a ring, for example where a phenyl group issubstituted with two occurrences of

these two occurrences of R^(o) are taken together with the oxygen atomsto which they are bound to form a fused 6-membered oxygen containingring:

It will be appreciated that a variety of other rings can be formed whentwo independent occurrences of R^(o) (or R⁺, or any other variablesimilarly defined herein) are taken together with the atom(s) to whicheach variable is bound and that the examples detailed above are notintended to be limiting.Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention. Additionally, unless otherwise stated, structuresdepicted herein are also meant to include compounds that differ only inthe presence of one or more isotopically enriched atoms. For example,compounds having the present structures except for the replacement ofhydrogen by deuterium or tritium, or the replacement of a carbon by a¹³C— or ¹⁴C-enriched carbon are within the scope of this invention. Suchcompounds are useful, for example, as analytical tools or probes inbiological assays.

3. Description of Exemplary Compounds

In some embodiments, R′ is independently selected from hydrogen or anoptionally substituted group selected from a C₁-C₈ aliphatic group, a3-8-membered saturated, partially unsaturated, or fully unsaturatedmonocyclic ring having 0-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or an 8-12 membered saturated, partiallyunsaturated, or fully unsaturated bicyclic ring system having 0-5heteroatoms independently selected from nitrogen, oxygen, or sulfur; ortwo occurrences of R′ are taken together with the atom(s) to which theyare bound to form an optionally substituted 3-12 membered saturated,partially unsaturated, or fully unsaturated monocyclic or bicyclic ringhaving 0-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur.

In some embodiments, each of Q, X, and W is independently a bond or isan optionally substituted C₁-C₆ alkylidene chain wherein up to twomethylene units of Q, W, or X are optionally and independently replacedby —CO—, —CS—, —COCO—, —CONR′—, —CONR′NR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—,—NR′CONR′—, —OCONR′—, —NR′NR′, —NR′NR′CO—, —NR′CO—, —S—, —SO, —SO₂—,—NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—.

In some embodiments, each of R^(X), R^(Q), and R^(W) is independentlyR′, halo, N₂, CN, CF₃, or OCF₃;

In other embodiments, Q is independently a bond or is an optionallysubstituted C₁₋₆ alkylidene chain wherein one or two non-adjacentmethylene units are optionally and independently replaced by O, NR, S,SO₂, COO, or CO, and R^(Q) is R′ or halogen. In still other embodiments,each occurrence of QR^(Q) is independently —C₁₋₃alkyl, —O(C₁₋₃alkyl),—CF₃, —OCF₃, —SCF₃, —F, —Cl, —Br, or —COOR′, —COR′, —O(CH₂)₂N(R)(R′),—O(CH₂)N(R)(R′), —CON(R)(R′), —(CH₂)₂OR′, —(CH₂)OR′, optionallysubstituted phenyl, —N(R)(R′), —(CH₂)₂N(R)(R′), or —(CH₂)N(R)(R′).

In other embodiments, X is independently a bond or is an optionallysubstituted C₁₋₆ alkylidene chain wherein one or two non-adjacentmethylene units are optionally and independently replaced by O, NR, S,SO₂, COO, or CO, and R^(X) is R′ or halogen. In still other embodiments,each occurrence of XR^(X) is independently —C₁₋₃alkyl, —O(C₁₋₃alkyl),—CF₃, —OCF₃, —SCF₃, —F, —Cl, —Br, or —COOR′, —COR′, —O(CH₂)₂N(R)(R′),—O(CH₂)N(R)(R′), —CON(R)(R′), —(CH₂)₂OR′, —(CH₂)OR′, optionallysubstituted phenyl, —N(R)(R′), —(CH₂)₂N(R)(R′), or —(CH₂)N(R)(R′). Inyet other embodiments, XR^(X) taken together is independently halo, OH,OMe, OEt, CH₂N(Me)₂, OCH₂CH₂OCH₃, CN, C(O)NH₂, Me, Et, NH₂, COOH, SO₂Me,N(Me)₂, NHC(O)CH₂N(Me)₂, pyrazolyl, N(Et)₂, OCF₃, SMe,C(O)—NH(4-methyl-oxazol-2-yl), C(O)NHCH₂CH₂OH, NHSO₂Me, N-morpholinyl,N(Me)-piperazinyl, or OC(O)NHEt.

In other embodiments, W is independently a bond or is an optionallysubstituted C₁₋₆ alkylidene chain wherein one or two non-adjacentmethylene units are optionally and independently replaced by O, NR, S,SO₂, COO, or CO, and R^(W) is R′ or halogen. In still other embodiments,each occurrence of WR^(W) is independently —C₁₋₃alkyl, —O(C₁₋₃alkyl),—CF₃, —OCF₃, —SCF₃, —F, —Cl, —Br, or —COOR′, —COR′, —O(CH₂)₂N(R)(R′),—O(CH₂)N(R)(R′), —CON(R)(R′), —(CH₂)₂OR′, —(CH₂)OR′, optionallysubstituted phenyl, —N(R)(R′), —(CH₂)₂N(R)(R′), or —(CH₂)N(R)(R′).

In one embodiment of formula I, R is hydrogen. In another embodiment offormula I, R^(N) is hydrogen.

In some embodiments, ring A is a 3-7 membered cycloalkyl ring.

In other embodiments, ring A is a 3-7 membered ring containing 1heteroatom selected from O, NR, or S. Or, ring A contains up twoheteroatoms selected from O, S, or NR.

In one embodiment, ring A is selected from:

In some embodiments, Ht is an optionally substituted 5-memberedheteroaromatic ring containing 1-4 heteroatoms selected from O, S, N, orNH, wherein said ring is optionally fused to a phenyl ring.

In certain embodiments, Ht is selected from one of the following rings:

wherein each ring is linked to the remainder of the molecule through acarbon ring atom.

In some embodiments, ring B is fused to 5-6 membered carbocyclic orheterocyclic, aromatic or non-aromatic ring. In certain embodiments,ring B is fused to a five membered heterocyclic ring.

In some embodiments, ring B is optionally substituted phenyl.

According to one embodiment, the present invention provides compounds offormula IIA:

wherein:

m is 0-4;

Ht₁ is a 5-membered heteroaromatic ring containing 1-4 heteroatomsselected from O, S, N, or NH, wherein said ring is optionally fused to aphenyl or 6-membered heteroaromatic ring;

ring B is optionally fused to 5-6 membered carbocyclic or heterocyclic,aromatic or non-aromatic ring;

X, R^(X), x, W, R^(W), and w are as defined above.

In some embodiments of formula IIA, m is 0. In other embodiments, mis 1. In yet other embodiments, m is 2. Or, m is 3. In certain otherembodiments, m is 4.

In certain embodiments of formula IIA, Ht¹ is an optionally substitutedring selected from:

wherein each ring is linked to the remainder of the molecule through acarbon ring atom.

Preferred Ht¹ include 1-a, 1-d, 1-f, 1-h, 1-i, 1-j, 1-l, 1-s-b, or 1-vrings above.

In certain embodiments of formula IIA, ring B is phenyl optionallysubstituted with up to x occurrences of X—R^(X). In other embodiments,ring B is fused with a 5 membered ring, such as, methylenedioxy,imidazolyl, or triazolyl.

In certain embodiments of formula IIA, x is 0-3. Preferably, x is 0-2.

According to another embodiment, the present invention providescompounds of formula IIIA or formula IIIB:

wherein:

m is 0 to 4;

Ar is phenyl or a six-membered heteroaromatic ring;

L is a bond, O, S, SO, SO₂, C(O), NR′, C₁₋₄ aliphatic, or CHR^(L);

R^(L) is —OR′, —SR′, —SOR′, —SO₂R′, or —N(R′)₂; or

wherein ring A′ is a 3-7 membered monocyclic ring having 0-3 heteroatomsselected from O, S, N, or NH, wherein ring A′ is optionally substitutedwith q occurrences of -QR^(Q);

R′ is as defined above;

X₉ is CH₂ or CF₂;

Ht₁ is a 5-membered heteroaromatic ring containing 1-4 heteroatomsselected from O, S, N, or NH, wherein said ring is optionally fused to aphenyl ring.

In certain embodiments of formula IIIA or formula IIIB, Ht¹ is anoptionally substituted ring selected from:

wherein each ring is linked to the remainder of the molecule through acarbon ring atom.

Preferred Ht¹ in formula IIIA or formula IIIB include a, h′, d, f, i, j,l, s-b, and v rings above.

In certain embodiments of formula IIIA or formula IIIB ring Ar isselected from:

In certain embodiments of formula IIIA or formula IIIB, m is 0. In otherembodiments thereof, m is 1. In yet other embodiments thereof, m is 2.Or, m is 3. In certain other embodiments thereof, m is 4.

According to another embodiment, the present invention providescompounds of formula IVA or formula IVB:

wherein:

L is a bond, O, S, SO, SO₂, C(O), NR′, C₁₋₄ aliphatic, or CHR^(L);

R^(L) is —OR′, —SR′, —SOR′, —SO₂R′, or —N(R′)₂; or

wherein ring A′ is a 3-7 membered monocyclic ring having 0-3 heteroatomsselected from O, S, N, or NH, wherein ring A′ is optionally substitutedwith q occurrences of -QR^(Q);

R′ is as defined above;

X₉ is CH₂ or CF₂;

X¹ is O, S, or NR;

R is hydrogen or C₁₋₄ aliphatic; and

each of X² and X³ is independently selected from CH or N.

In another embodiment of formula IVA or formula IVB, L is C1-C4alkylidene wherein up to two carbon atoms are optionally replaced by O,S, or NR.

According to one embodiment of the present invention, L is —CH₂—, thusproviding compounds of formula IVA-1 or formula IVB-1:

According to one embodiment of formula IVA-1 or formula IVB-1, X¹ is S,X² is N, and X³ is CH.

According to another embodiment of formula IVA-1 or formula IVB-1, X¹ isS, X² and X³ both are N.

According to one embodiment of formula IVA-1 or formula IVB-1, X¹ is S,X² is CH, and X³ is N.

According to one embodiment of formula IVA-1 or formula IVB-1, X¹ is O,X² is N, and X³ is CH.

According to another embodiment of formula IVA-1 or formula IVB-1, X¹ isO, X² and X³ both are N.

According to one embodiment of formula IVA-1 or formula IVB-1, X¹ is O,X² is CH, and X³ is N.

According to one embodiment of formula IVA-1 or formula IVB-1, X¹ is NR,X² is N, and X³ is CH. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to another embodiment of formula IVA-1 or formula IVB-1, X¹ isNR, X² and X³ both are N. In one embodiment, R is hydrogen. Or, R isC₁₋₄ alkyl.

According to one embodiment of formula IVA-1 or formula IVB-1, X¹ is NR,X² is CH, and X³ is N. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to one embodiment of the present invention, L is —C(O), thusproviding compounds of formula IVA-2 or formula IVB-1:

According to one embodiment of formula IVA-2 or formula IVB-2, X¹ is S,X² is N, and X³ is CH.

According to another embodiment of formula IVA-2 or formula IVB-2, X¹ isS, X² and X³ both are N.

According to one embodiment of formula IVA-2 or formula IVB-2, X¹ is S,X² is CH, and X³ is N.

According to one embodiment of formula IVA-2 or formula IVB-2, X¹ is O,X² is N, and X³ is CH.

According to another embodiment of formula IVA-2 or formula IVB-2, X¹ isO, X² and X³ both are N.

According to one embodiment of formula IVA-2 or formula IVB-2, X¹ is O,X² is CH, and X³ is N.

According to one embodiment of formula IVA-2 or formula IVB-2, X¹ is NR,X² is N, and X³ is CH. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to another embodiment of formula IVA-2 or formula IVB-2, X¹ isNR, X² and X³ both are N. In one embodiment, R is hydrogen. Or, R isC₁₋₄ alkyl.

According to one embodiment of formula IVA-2 or formula IVB-2, X¹ is NR,X² is CH, and X³ is N. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to another embodiment, L is S, SO, or SO₂, thus providingcompounds of formula IVA-3 or formula IVB-3:

wherein L is S, SO, or SO₂.

According to one embodiment of formula IVA-3 or formula IVB-3, X¹ is S,X² is N, and X³ is CH.

According to another embodiment of formula IVA-3 or formula IVB-3, X¹ isS, X² and X³ both are N.

According to one embodiment of formula IVA-3 or formula IVB-3, X¹ is S,X² is CH, and X³ is N.

According to one embodiment of formula IVA-3 or formula IVB-3, X¹ is O,X² is N, and X³ is CH.

According to another embodiment of formula IVA-3 or formula IVB-3, X¹ isO, X² and X³ both are N.

According to one embodiment of formula IVA-3 or formula IVB-3, X¹ is O,X² is CH, and X³ is N.

According to one embodiment of formula IVA-3 or formula IVB-3, X¹ is NR,X² is N, and X³ is CH. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to another embodiment of formula IVA-3 or formula IVB-3, X¹ isNR, X² and X³ both are N. In one embodiment, R is hydrogen. Or, R isC₁₋₄ alkyl.

According to one embodiment of formula IVA-3 or formula IVB-3, X¹ is NR,X² is CH, and X³ is N. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to another embodiment, L is O, thus providing compounds offormula IVA-4 or formula IVD-4:

According to one embodiment of formula IVA-4 or formula IVB-4, X¹ is S,X² is N, and X³ is CH.

According to another embodiment of formula IVA-4 or formula IVB-4, X¹ isS, X² and X³ both are N.

According to one embodiment of formula IVA-4 or formula IVB-4, X¹ is S,X² is CH, and X³ is N.

According to one embodiment of formula IVA-4 or formula IVB-4, X¹ is O,X² is N, and X³ is CH.

According to another embodiment of formula IVA-4 or formula IVB-4, X¹ isO, X² and X³ both are N.

According to one embodiment of formula IVA-4 or formula IVB-4, X¹ is O,X² is CH, and X³ is N.

According to one embodiment of formula IVA-4 or formula IVB-4, X¹ is NR,X² is N, and X³ is CH. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to another embodiment of formula IVA-4 or formula IVB-4, X¹ isNR, X² and X³ both are N. In one embodiment, R is hydrogen. Or, R isC₁₋₄ alkyl.

According to one embodiment of formula IVA-4 or formula IVB-4, X¹ is NR,X² is CH, and X³ is N. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to another embodiment, L is a bond, thus providing compoundsof formula IVA-5 or formula IVA-6:

According to one embodiment of formula IVA-5 or IVB-5, X¹ is S, X² is N,and X³ is CH.

According to another embodiment of formula IVA-5 or IVB-5, X¹ is S, X²and X³ both are N.

According to one embodiment of formula IVA-5 or IVB-5, X¹ is S, X² isCH, and X³ is N.

According to one embodiment of formula IVA-5 or IVB-5, X¹ is O, X² is N,and X³ is CH.

According to another embodiment of formula IVA-5 or IVB-5, X¹ is O, X²and X³ both are N

According to one embodiment of formula IVA-5 or IVB-5, X¹ is O, X² isCH, and X³ is N.

According to one embodiment of formula IVA-5 or IVB-5, X¹ is NR, X² isN, and X³ is CH. In one embodiment, R is hydrogen. Or, R is C₁₋₄ alkyl.

According to another embodiment of formula IVA-5 or IVB-5, X¹ is NR, X²and X³ both are N. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to one embodiment of formula IVA-5 or IVB-5, X¹ is NR, X² isCH, and X³ is N. In one embodiment, R is hydrogen. Or, R is C₁₋₄ alkyl.

According to one embodiment of formula IVA-6 or IVB-6, X¹ is S, X² is N,and X³ is CH.

According to one embodiment of formula IVA-6 or IVB-6, X¹ is O, X² is N,and X³ is CH.

According to one embodiment of formula IVA-6 or IVB-6, X¹ is O, X² isCH, and X³ is N.

According to another embodiment, L is —NR′—, thus providing compounds offormula IVA-7 or formula IVB-7:

wherein:

R′ is hydrogen or R^(D);

R^(D) is C₁₋₆ aliphatic optionally substituted with —OH,—O(C₁₋₄aliphatic), —S(C₁₋₄aliphatic), —CF₃, —OCF₃, —SCF₃, halo, NH₂,NHR, N(R)₂, C(O)OH, C(O)O(C₁₋₄aliphatic), NHC(O)(C₁₋₄aliphatic), or a3-7 membered heterocyclic ring containing up to 4 heteroatoms selectedfrom O, N, or S, wherein said ring is optionally substituted with up to2 substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y;

p is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OR, SR, NH₂, NHR, N(R)₂, COOH, or COOR;

R is hydrogen or C₁₋₄ aliphatic;

According to one embodiment of formula IVA-7 or formula IVB-7, X¹ is S,X² is N, and X³ is CH.

According to another embodiment of formula IVA-7 or formula IVB-7, X¹ isS, X² and X³ both are N.

According to one embodiment of formula IVA-7 or formula IVB-7, X¹ is S,X² is CH, and X³ is N.

According to one embodiment of formula IVA-7 or formula IVB-7, X¹ is O,X² is N, and X³ is CH.

According to another embodiment of formula IVA-7 or formula IVB-7, X¹ isO, X² and X³ both are N.

According to one embodiment of formula IVA-7 or formula IVB-7, X¹ is O,X² is CH, and X³ is N.

According to one embodiment of formula IVA-7 or formula IVB-7, X¹ is NR,X² is N, and X³ is CH. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to another embodiment of formula IVA-7 or formula IVB-7, X¹ isNR, X² and X³ both are N. In one embodiment, R is hydrogen. Or, R isC₁₋₄ alkyl.

According to one embodiment of formula IVA-7 or formula IVB-7, X¹ is NR,X² is CH, and X³ is N. In one embodiment, R is hydrogen. Or, R is C₁₋₄alkyl.

According to another embodiment, the present invention providescompounds of formula VA-1, formula VA-2, formula VA-3, or formula VA-4:

wherein:

each of R^(AA), R^(BB), R^(C), R^(D), and R^(E) is independentlyhydrogen or C₁₋₆ aliphatic optionally substituted with —OH,—O(C₁₋₄aliphatic), —S(C₁₋₄aliphatic), —CF₃, —OCF₃, —SCF₃, halo, NH₂,NHR, N(R)₂, C(O)OH, C(O)O(C₁₋₄aliphatic), NHC(O)(C₁₋₄aliphatic), or a3-7 membered heterocyclic ring containing up to 4 heteroatoms selectedfrom O, N, or S, wherein said ring is optionally substituted with up to2 substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y;

p is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OR, SR, NH₂, NHR, N(R)₂, COOH, or COOR;

R is hydrogen or C₁₋₄ aliphatic;

R^(AA) and R^(BB), taken together with the nitrogen atom, is a 3-7membered heterocyclic ring containing up to 4 heteroatoms selected fromO, N, or S, wherein said ring is optionally substituted with up to 2substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y;

p is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OR, SR, NH₂, NHR, N(R)₂, COOH, or COOR;and

R is hydrogen or C₁₋₄ aliphatic.

In certain embodiments, R^(C) is C₁₋₄ alkylidene optionally substitutedwith up to two substituents selected from —OH, —O(C₁₋₄alkyl), NH(C₁₋₄alkyl), or N(C₁₋₄ alkyl)₂. In other embodiments, R^(C) is C₁₋₄alkylidene optionally substituted with 5-6 membered heterocyclic ringcontaining up to 2 heteroatoms selected from O, N, or S, wherein saidring is optionally substituted with up to 2 substituents selected fromoxo, (C₁₋₄ aliphatic), (C₁₋₄ aliphatic)-Y, wherein Y is halo, —OH, or—O(C₁₋₄ alkyl).

Preferred embodiments of R^(C) in the present invention includehydrogen, methoxy, ethoxy, —(CH₂)₂-(4-hydroxy-1-piperidyl),—(CH₂)₃-(4-hydroxy-1-piperidyl), —(CH₂)₄-(4-hydroxy-1-piperidyl),ethylmethylamino-ethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl,2-methoxyethyl, isopropyl, (2-methoxymethyl-1-pyrrolidinyl)ethyl,tetrahydrofuran-2-ylmethyl, tetrahydrofuran-3-ylmethyl,(pyrrolidin-2-one-5-yl)methyl, (pyrrolidin-1-yl)ethyl,dimethylaminoethyl, 1-piperidylethyl, allyl, n-propyl,diisopropylaminoethyl, and (N-morpholino)ethyl.

In one embodiment of formula VA-1:

a. z is 0-2 and ZR^(Z) together is selected from halo, OMe, OEt, CN,C(O)NH₂, Me, Et, NH₂, COOH, SO₂Me, N(Me)₂, or N(Et)₂;

b. x is 0-2 and XR^(X) together is selected from halo, OMe, OEt, CN,C(O)NH₂, Me, Et, NH₂, COOH, SO₂Me, N(Me)₂, N(Et)₂, OCF₃, or SMe; and

c. R^(C) is hydrogen, —(CH₂)₂-(4-hydroxy-1-piperidyl),—(CH₂)₃-(4-hydroxy-1-piperidyl), —(CH₂)₄-(4-hydroxy-1-piperidyl),(ethylmethylamino)ethyl, 2-hydroxyethyl, 3-hydroxypropyl,4-hydroxybutyl, 2-methoxyethyl, isopropyl,(2-methoxymethyl-1-pyrrolidinyl)ethyl, tetrahydrofuran-2-ylmethyl,tetrahydrofuran-3-ylmethyl, (pyrrolidin-2-one-5-yl)methyl,(pyrrolidin-1-yl)ethyl, dimethylaminoethyl, 1-piperidylethyl, allyl,n-propyl, diisopropylaminoethyl, or (N-morpholino)ethyl.

In certain embodiments, R^(D) is C₁₋₄ alkylidene optionally substitutedwith —OH, —O(C₁₋₄alkyl), NH(C₁₋₄ alkyl), or N(C₁₋₄ alkyl)₂. In otherembodiments, R^(D) is C₁₋₄ alkylidene optionally substituted with 5-6membered heterocyclic ring containing up to 2 heteroatoms selected fromO, N, or S, wherein said ring is optionally substituted with up to 2substituents selected from oxo, (C₁₋₄ aliphatic), (C₁₋₄ aliphatic)-Y,wherein Y is halo, —OH, or —O(C₁₋₄ alkyl).

Preferred embodiments of R^(D) include (N-morpholino)ethyl,(N-morpholino)propyl, dimethylaminoethyl, or (N-piperidyl)ethyl.

In one embodiment of formula VA-2:

a. z is 0-2 and ZR^(Z) together is selected from halo, OMe, OEt, CN,C(O)NH₂, Me, Et, NH₂, COOH, SO₂Me, N(Me)₂, or N(Et)₂;

b. x is 0-2 and XR^(X) together is selected from halo, OMe, OEt, CN,C(O)NH₂, Me, Et, NH₂, COOH, SO₂Me, N(Me)₂, N(Et)₂, OCF₃, or SMe; and

c. R^(D) is (N-morpholino)ethyl, (N-morpholino)propyl,dimethylaminoethyl, or (N-piperidyl)ethyl.

In certain embodiments of formula VA-3, each of R^(AA) and R^(BB) isindependently C₁₋₄ alkylidene optionally substituted with up to twosubstituents selected from —OH, —O(C₁₋₄alkyl), NH(C₁₋₄ alkyl), or N(C₁₋₄alkyl)₂. In other embodiments, R^(AA) and R^(BB) is C₁₋₄ alkylideneoptionally substituted with 5-6 membered heterocyclic ring containing upto 2 heteroatoms selected from O, N, or S, wherein said ring isoptionally substituted with up to 2 substituents selected from oxo,(C₁₋₄ aliphatic), (C₁₋₄ aliphatic)-Y, wherein Y is halo, —OH, or —O(C₁₋₄alkyl).

Preferred R^(AA) and R^(BB) include Me, Et, propyl, butyl, allyl,hydroxyethyl, dihydroxypropyl, or diisopropylaminoethyl.

In certain embodiments, R^(AA) and R^(BB), taken together with thenitrogen atom, is selected from:

Preferred R^(AB) include methoxymethyl, methoxyethyl, allyl, methyl,—OH, hydroxymethyl, hydroxyethyl, ethylenedioxy, COOH, CONH₂, orC(O)CH₃.

According to one embodiment of formula VA-4, R^(E) is independentlyhydrogen or C₁₋₆ aliphatic optionally substituted with —OH,—O(C₁₋₄aliphatic), —S(C₁₋₄aliphatic), —CF₃, —OCF₃, —SCF₃, halo, NH₂,NHR, N(R)₂, C(O)OH, C(O)O(C₁₋₄aliphatic), or NHC(O)(C₁₋₄aliphatic).Preferred embodiments of R^(E) include —(CH₂)₂OH, —(CH₂)₃OH, —(CH₂)₄OH,—(CH₂)₂NH₂, —(CH₂)₂NMe₂, —(CH₂)₂NEt₂, —(CH₂)₂NHC(O)Me, —(CH₂)COOH,—(CH₂)₂COOH, —(CH₂)COOMe, —(CH₂)CH(NH₂)COOH, or —(CH₂)₂CH(NH₂)COOH.

According to another embodiment, the present invention providescompounds of formula VB-1, formula VB-2, formula VB-3, of formula VB-4:

wherein:

each of R^(AA), R^(BB), R^(C), R^(D) and R^(D) is independently hydrogenor C₁₋₆ aliphatic optionally substituted with —O(C₁₋₄aliphatic), —CF₃,—OCF₃, —SCF₃, halo, NH₂, NHR, N(R)₂, or a 3-7 membered heterocyclic ringcontaining up to 4 heteroatoms selected from O, N, or S, wherein saidring is optionally substituted with up to 2 substituents selected fromoxo or (C₁₋₄aliphatic)_(p)-Y;

p is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OR, SR, NH₂, NHR, N(R)₂, COOH, or COOR;

R is hydrogen or C₁₋₄ aliphatic;

R^(AA) and R^(BB), taken together with the nitrogen atom, is a 3-7membered heterocyclic ring containing up to 4 heteroatoms selected fromO, N, or S, wherein said ring is optionally substituted with up to 2substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y;

p is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OR, SR, NH₂, NHR, N(R)₂, COOH, or COOR;and

R is hydrogen or C₁₋₄ aliphatic.

According to another embodiment, the present invention providescompounds of formula VIA or formula VIB:

wherein:

X¹ is O, S, or NR;

R is hydrogen or C₁₋₄ alkyl; and

X₉ is CH₂ or CF₂.

According to one embodiment of formula VIA, X¹ is S. Or, X¹ is O. Or, X¹is NR. In one embodiment, R is hydrogen. Or, R is C₁₋₄ alkyl.

According to one embodiment of formula VIB, X¹ is S. Or, X¹ is O. Or, X¹is NR. In one embodiment, R is hydrogen. Or, R is C₁₋₄ alkyl.

According to another embodiment, the present invention providescompounds of formula VIIA or formula VIIB:

wherein:

m is 0-4;

X₈ is O or S;

X₉ is CH₂ or CF₂; and

R″ is C₁₋₄ alkyl optionally substituted with NH(C₁₋₄ alkyl), N(C₁₋₄alkyl)₂, or C(O)O—(C₁₋₄ alkyl).

In certain embodiments of formula VIIA or formula VIIB, m is 0. Or, m is2 or 3.

In certain embodiments of formula VIIA or formula VIIB, R″ is methyl,CH₂—C(O)OMe, or CH₂CH₂—N(Et)₂.

In certain embodiments of formula VIIA or formula VIIB, X₈ is S. Or, X₈is O.

Exemplary compounds of the present invention are recited in below inTable 1. TABLE 1 Cmpd # Compound 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

4. General Synthetic Schemes

Compounds of formula I can be prepared by well-known methods in the art.Illustrated below are exemplary methods for the preparation of compoundsof formula I. Scheme I below illustrates an exemplary synthetic methodfor compounds of formula I.

General Procedure: One equivalent of the appropriate carboxylic acid andone equivalent of the appropriate amine were dissolved in acetonitrilecontaining triethylamine (3 equivalents).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) was added and the solution was allowed tostir. The crude product was purified by reverse-phase preparative liquidchromatography to yield the pure product.

5. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

As discussed above, the present invention provides compounds that areuseful as modulators of ABC transporters and thus are useful in thetreatment of disease, disorders or conditions such as Cystic fibrosis,Hereditary emphysema, Hereditary hemochromatosis,Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency,Type 1 hereditary angioedema, Lipid processing deficiencies, such asFamilial hypercholesterolemia, Type 1 chylomicronemia,Abetalipoproteinemia, Lysosomal storage diseases, such as I-celldisease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, Polyendocrinopathy/Hyperinsulemia, Diabetesmellitus, Laron dwarfism, Myleoperoxidase deficiency, Primaryhypoparathyroidism, Melanoma, Glycanosis CDG type 1, Hereditaryemphysema, Congenital hyperthyroidism, Osteogenesis imperfecta,Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders asuch as Huntington, Spinocerebullar ataxia typeI, Spinal and bulbar muscular atrophy, Dentatorubal pallidoluysian, andMyotonic dystrophy, as well as Spongiform encephalopathies, such asHereditary Creutzfeldt-Jakob disease (due to Prion protein processingdefect), Fabry disease and Straussler-Scheinker syndrome.

Accordingly, in another aspect of the present invention,pharmaceutically acceptable compositions are provided, wherein thesecompositions comprise any of the compounds as described herein, andoptionally comprise a pharmaceutically acceptable carrier, adjuvant orvehicle. In certain embodiments, these compositions optionally furthercomprise one or more additional therapeutic agents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative thereof. According to thepresent invention, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or any other adduct or derivative which uponadministration to a patient in need is capable of providing, directly orindirectly, a compound as otherwise described herein, or a metabolite orresidue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgement,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof. As used herein, the term “inhibitorily activemetabolite or residue thereof” means that a metabolite or residuethereof is also an inhibitor of an ATP-Binding Cassette Transporters.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersable products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating a condition, disease, or disorder implicated by ABC transporteractivity. In certain embodiments, the present invention provides amethod of treating a condition, disease, or disorder implicated by adeficiency of ABC transporter activity, the method comprisingadministering a composition comprising a compound of formula (I) to asubject, preferably a mammal, in need thereof.

In certain preferred embodiments, the present invention provides amethod of treating cystic fibrosis, hereditary emphysema (due toa1-antitrypsin; non Piz variants), hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses (due to lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are glycanosis CDGtype 1, hereditary emphysema (due to α1-antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-antichymotrypsin), diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A), Straussler-Scheinkersyndrome, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome, comprising the step of administering to saidmammal an effective amount of a composition comprising a compound offormula (I), or a preferred embodiment thereof as set forth above.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis comprising the step ofadministering to said mammal a composition comprising the step ofadministering to said mammal an effective amount of a compositioncomprising a compound of formula (I), or a preferred embodiment thereofas set forth above.

According to the invention an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective fortreating or lessening the severity of one or more of cystic fibrosis,hereditary emphysema (due to a1-antitrypsin; non Piz variants),hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, suchas protein C deficiency, Type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, Type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses (due to lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are glycanosis CDGtype 1, hereditary emphysema (due to α1-antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-antichymotrypsin), diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to ΔAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A), Straussler-Scheinkersyndrome, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of oneor more of cystic fibrosis, hereditary emphysema (due to a1-antitrypsin;non Piz variants), hereditary hemochromatosis, coagulation-fibrinolysisdeficiencies, such as protein C deficiency, Type 1 hereditaryangioedema, lipid processing deficiencies, such as familialhypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,lysosomal storage diseases, such as I-cell disease/pseudo-Hurler,mucopolysaccharidoses (due to lysosomal processing enzymes),Sandhof/Tay-Sachs (due to β-hexosaminidase), Crigler-Najjar type II (dueto UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are glycanosis CDGtype 1, hereditary emphysema (due to α1-antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-antichymotrypsin), diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A), Straussler-Scheinkersyndrome, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar—agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are prepared by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

As described generally above, the compounds of the invention are usefulas modulators of ABC transporters. Thus, without wishing to be bound byany particular theory, the compounds and compositions are particularlyuseful for treating or lessening the severity of a disease, condition,or disorder where hyperactivity or inactivity of ABC transporters isimplicated in the disease, condition, or disorder. When hyperactivity orinactivity of an ABC transporter is implicated in a particular disease,condition, or disorder, the disease, condition, or disorder may also bereferred to as a “ABC transporter-mediated disease, condition ordisorder”. Accordingly, in another aspect, the present inventionprovides a method for treating or lessening the severity of a disease,condition, or disorder where hyperactivity or inactivity of an ABCtransporter is implicated in the disease state.

The activity of a compound utilized in this invention as a modulator ofan ABC transporter may be assayed according to methods describedgenerally in the art and in the Examples herein.

It will also be appreciated that the compounds and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the compounds and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another agent used to treat the same disorder), orthey may achieve different effects (e.g., control of any adverseeffects). As used herein, additional therapeutic agents that arenormally administered to treat or prevent a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated”.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating an implantable medical device, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present invention includes an implantable device coated witha composition comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

Another aspect of the invention relates to modulating ABC transporteractivity in a biological sample or a patient (e.g., in vitro or invivo), which method comprises administering to the patient, orcontacting said biological sample with a compound of formula I or acomposition comprising said compound. The term “biological sample”, asused herein, includes, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof.

Modulation of ABC transporter activity in a biological sample is usefulfor a variety of purposes that are known to one of skill in the art.Examples of such purposes include, but are not limited to, the study ofABC transporters in biological and pathological phenomena; and thecomparative evaluation of new modulators of ABC transporters.

In yet another embodiment, a method of modulating activity of an anionchannel in vitro or in vivo, is provided comprising the step ofcontacting said channel with a compound of formula (I). In preferredembodiments, the anion channel is a chloride channel or a bicarbonatechannel. In other preferred embodiments, the anion channel is a chloridechannel.

According to an alternative embodiment, the present invention provides amethod of increasing the number of functional ABC transporters in amembrane of a cell, comprising the step of contacting said cell with acompound of formula (I). The term “functional ABC transporter” as usedherein means an ABC transporter that is capable of transport activity.In preferred embodiments, said functional ABC transporter is CFTR.

According to another preferred embodiment, the activity of the ABCtransporter is measured by measuring the transmembrane voltagepotential. Means for measuring the voltage potential across a membranein the biological sample may employ any of the known methods in the art,such as optical membrane potential assay or other electrophysiologicalmethods.

The optical membrane potential assay utilizes voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission can be monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

In another aspect the present invention provides a kit for use inmeasuring the activity of a ABC transporter or a fragment thereof in abiological sample in vitro or in vivo comprising (i) a compositioncomprising a compound of formula (I) or any of the above embodiments;and (ii) instructions for a) contacting the composition with thebiological sample and b) measuring activity of said ABC transporter or afragment thereof. In one embodiment, the kit further comprisesinstructions for a) contacting an additional composition with thebiological sample; b) measuring the activity of said ABC transporter ora fragment thereof in the presence of said additional compound, and c)comparing the activity of the ABC transporter in the presence of theadditional compound with the density of the ABC transporter in thepresence of a composition of formula (I). In preferred embodiments, thekit is used to measure the density of CFTR.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES Example 1

2-Bromo-1-(chloro-phenyl)-ethanone Bromine (3.8 mL, 65 mmol) was addeddropwise to a solution of 1-(2-chloro-phenyl)-ethanone (10. g, 65 mmol)in acetic acid (75 mL) at 0° C. The mixture was then warmed to roomtemperature and stirred overnight. The mixture was evaporated to drynessand used in the next step without further purification.N′-[5-(2-Chloro-benzoyl)-thiazol-2-yl]-N,N-dimethyl-formamidine. Amixture of thiourea (4.95 g, 65.0 mmol) anddimethoxymethyl-dimethyl-amine (23.2 g, 195 mmol) in methanol (80 mL)was heated under reflux for 30 minutes. After allowing the mixture tocool, triethylamine (19.8 g, 195 mmol) and a solution of2-bromo-1-(chloro-phenyl)-ethanone (crude from last step) in methanol(50 mL) were added. The mixture was heated under reflux for 4 hours. Thesolvent was removed and the residue was used directly in the nextprocedure.

(2-Amino-thiazol-5-yl)-(2-chloro-phenyl)-methanone The crudeN′-[5-(2-chloro-benzoyl)-thiazol-2-yl]-N,N-dimethyl-formamidine wasdissolved in 10% hydrochloric acid (150 mL) and heated to 70° C. for 4hours. The precipitate was filtered, washed with ether, and thensuspended in a 10% sodium carbonate solution (250 mL). The suspensionwas stirred for 1 hour and the precipitate was filtered, washed withether, and dried in air to give(2-amino-thiazol-5-yl)-(2-chloro-phenyl)-methanone as a brown solid (8.5g, 36 mmol, 55% from 1-(2-chloro-phenyl)-ethanone). ESI-MS m/z calc.238.0, found; 239.3 (M+1)⁺ ¹H NMR (DMSO): δ: 7.252 (s, 1 H), 7.420-7.553(m, 4 H), 8.345 (s, 2 H).

(2-Amino-thiazol-5-yl)-(2-methoxy-phenyl)-methanone(2-Amino-thiazol-5-yl)-(2-methoxy-phenyl)-methanone was prepared in amanner analogous to that of(2-amino-thiazol-5-yl)-(2-chloro-phenyl)-methanone (92% yield). ESI-MSm/z calc. 234.1, found; 235.1 (M+1)⁺ ¹H NMR (CDCl₃): δ: 7.37-7.7.47 (m,3 H), 6.98-7.04 (m, 2 H), 5.77 (br, 1 H), 3.82 (s, 3 H).

2-Chloro-3-(2-chloro-phenyl)-propionaldehyde To a solution of2-chloroaniline (12.7 g, 100. mmol) in hydrochloric acid (20% aqueoussolution, 40 mL) was added dropwise a solution of sodium nitrite (7.5 g,110 mmol) in water (20 mL) at 0 to 5° C. After stirring for 10 minutes,a cooled solution of acrolin (15 g, 270 mmol) in acetone (100 mL)containing calcium oxide (2.0 g, 36 mmol) was added gradually, and thenfollowed by a solution of cuprous chloride (1 g, 10 mmol) in acetone (10mL) containing hydrochloric acid (20% aqueous solution, 2 mL). Themixture was stirred at 0 to 30° C. for 3 hours and then extracted threetimes with dichloromethane (100 mL). The combined organic layers werewashed with a saturated aqueous solution of sodium bicarbonate followedby a saturated aqueous solution of sodium chloride. The organic layerwas separated, dried over sodium sulfate, and evaporated to dryness togive a black viscous oil. The crude product was passed through a shortsilica gel column to give 12 g of crude product, which was used directlyin the next step.

5-(2-Chloro-benzyl)-thiazol-2-ylamine A mixture of2-chloro-3-(2-chloro-phenyl)-propionaldehyde (12 g, crude from above)and urea (6.0 g, 0.10 mol) in ethanol (120 mL) was heated to refluxovernight. The solvent was evaporated to dryness. The residue wasdiluted with dichloromethane (120 mL) and then washed with sodiumhydroxide (10% aqueous solution, 50 mL ) and water (30 mL). The organiclayer was extracted three times with hydrochloric acid (5% aqueoussolution, 120 mL). The combined aqueous layer was adjusted with a 10%aqueous solution of sodium hydroxide to between pH 9 and 10 and thenextracted three times with dichloromethane (150 mL). The organic layerswere combined, dried over sodium sulfate, evaporated to dryness, andpurified by silica gel column chromatography to yield a yellow solid.(5.2 g, 0.023 mol, 23% from 2-chloroaniline). ESI-MS m/z calc. 224.0,found 225.2 (M+1)⁺ ¹H NMR (CDCl₃) δ 4.07 (s, 2H), 4.90 (bs, 2H), 6.80(s, 1H), 7.37-7.15 (m, 4H).

5-(2-methoxy-benzyl)-thioazol-2-ylamine5-(2-methoxy-benzyl)-thioazol-2-ylamine was prepared in a manneranalogous to that of 5-(2-chloro-benzyl)-thiazol-2-ylamine. ESI-MS m/zcalc. 220.1, found 221.2 (M+1)⁺ ¹H NMR(CDCl₃) δ 7.26-7.19 (m, 1H), 7.15(d, J=6.8 Hz, 1H), 6.90-6.85 (m, 2H), 6.79 (s, 1H), 4.77 (bs, 2H), 3.93(s, 2H), 3.84 (s, 3H).

5-(3-Chloro-benzyl)-thioxazol-2-ylamine5-(3-Chloro-benzyl)-thioxazol-2-ylamine was prepared in a manneranalogous to that of 5-(2-chloro-benzyl)-thiazol-2-ylamine. ESI-MS m/zcalc. 224.0, found 225.2 (M+1)⁺ ¹H NMR (CDCl₃) δ 7.26-7.21 (m, 3H), 7.10(d, J=6.8 Hz, 1H), 6.81 (s, 1H), 4.82 (bs, 2H), 3.93 (s, 2H).

5-(4-Chloro-benzyl)-thioxazol-2-ylamine5-(4-Chloro-benzyl)-thioxazol-2-ylamine was prepared in a manneranalogous to that of 5-(2-chloro-benzyl)-thiazol-2-ylamine. ESI-MS m/zcalc. 224.0, found 225.2 (M+1)⁺ ¹H NMR(CDCl₃) δ 7.26 (d, J=8.4 Hz, 2H),7.14 (d, J=8.4 Hz, 2H), 6.79 (s, 1H), 4.85 (bs, 2H), 3.92 (s, 2H).

5-(2-Cyano-benzyl)-thiazol-2-ylamine5-(2-Cyano-benzyl)-thiazol-2-ylamine was prepared in a manner analogousto that of 5-(2-chloro-benzyl)-thiazol-2-ylamine (12 g, 56 mmol, 11%from 2-cyanoaniline). ESI-MS m/z calc. 215.05, found 216.16 (M+1)⁺ ¹HNMR(CDCl₃): δ 7.64 (d, 1H), 7.54 (t, 1H), 7.34 (m, 2H), 6.87 (s, 1H),4.89 (br, 2H), 4.19 (s, 2H).

5-(2-Chloro-3-fluoro-benzyl)-thiazol-2-ylamine5-(2-Chloro-3-fluoro-benzyl)-thiazol-2-ylamine was prepared in a manneranalogous to that of 5-(2-chloro-benzyl)-thiazol-2-ylamine (4.0 g, 16mmol, 12% from 2-chloro-6-fluoroaniline). ESI-MS m/z calc. 242.07, found243.14 (M+1)⁺ ¹H NMR(CDCl₃): δ 7.17 (m, 1H), 7.04 (m, 2H), 6.82 (s, 1H),4.86 (br, 2H), 4.09 (s, 2H).

5-(2-chloro-4-fluoro-benzyl)-thiazol-2-ylamine5-(2-chloro-4-fluoro-benzyl)-thiazol-2-ylamine was prepared in a manneranalogous to that of 5-(2-chloro-benzyl)-thiazol-2-ylamine (11.5 g, 47.4mmol, 30% from 2-chloro-4-fluoroaniline). ESI-MS m/z calc. 242.01, found243.27 (M+1)⁺ ¹H NMR(CDCl₃): δ 9.34 (br, 2 H), 7.48 (m, 2 H), 7.24 (m, 1H), 7.07 (s, 1 H), 4.04 (s, 2 H).

5-(2-chloro-5-fluoro-benzyl)-thiazol-2-ylamine5-(2-chloro-5-fluoro-benzyl)-thiazol-2-ylamine was prepared in a manneranalogous to that of 5-(2-chloro-benzyl)-thiazol-2-ylamine(7.8 g, 32mmol, 20% from 2-chloro-5-fluoroaniline). ESI-MS m/z calc. 242.01, found243.36 (M+1)⁺ ¹H NMR: (DMSO-d₆) δ 9.25 (s, 2H), 7.52 (q, 1H), 7.36 (dd,1H), 7.21 (dt, 1H), 7.10 (s, 1H), 4.05 (s, 2H).

Example 2

2-Chloro-3-(2-methoxy-phenyl)-propionaldehyde. A solution of2-methoxylaniline (24.6 g, 0.200 mole) in hydrochloric acid (20% aqueoussolution, 80 mL) was slowly added to a solution of sodium nitrite (15 g,0.22 mole) in water (40 mL) at 0 to 5° C. After stirring for 10 minutes,a cooled solution of acrolin (30 g, 0.56 mol) in acetone (200 mL)containing calcium oxide (4.0 g, 72 mmol) was added gradually, and thenfollowed by a solution of cuprous chloride (2.0 g, 20 mmol) in acetone(20 mL) containing hydrochloric acid (20% aqueous solution, 4 mL). Themixture was stirred at 0 to 30° C. for 3 hours, and then extracted withthree 150 mL portions of dichloromethane. The combined organic layerswere washed with a saturated aqueous solution of sodium bicarbonate, asaturated aqueous solution of sodium chloride, dried over sodiumsulfate, filtered, and concentrated to give a black viscous oil. Thecrude product was passed through a short silica column to give 10 g ofcrude product, which was used directly in the next procedure.

5-(2-methoxy-benzyl)-oxazol-2-ylamine. A mixture of2-chloro-3-(2-methoxylphenyl)-propionaldehyde (10 g, crude from above)and urea (9.6 g, 0.16 mol) was dissolved in ethanol (250 mL) and thenheated to reflux overnight. The solvent was evaporated to dryness. Theresidue was diluted with dichloromethane (250 mL) and then washed withsodium hydroxide (10% aqueous solution, 100 mL) and water (50 mL). Theorganic layer was extracted three times with hydrochloric acid (5%aqueous solution, 250 mL). The combined aqueous layers were adjusted topH 9 to 10 with a 10% aqueous solution of sodium hydroxide and thenextracted three times with dichloromethane (300 mL). The organic layerwas separated, dried over sodium sulfate, and evaporated to dryness. Thecrude product was purified by silica gel column chromatography to yieldthe yellow-red solid product. (0.72 g, 0.35% from 2-methoxyaniline).ESI-MS m/z calc. 204.1, found; 205.1 (M+1)⁺ ¹H NMR(CDCl₃) δ 7.26-7.20(m, 1H), 7.14 (d, J=7.2 Hz, 1H), 6.91-6.86 (m, 2H), 6.35 (s, 1H), 4.49(bs, 2H), 3.85 (s, 2H), 3.82 (s, 3H).

5-(2-Chloro-benzyl)-oxazol-2-ylamine.5-(2-Chloro-benzyl)-oxazol-2-ylamine was prepared in a manner analogousto that of the preparation of 5-(2-methoxy-benzyl)-oxazol-2-ylamine toyield the product as a yellow solid. (3.5 g, 8.4% from 2-chloroaniline).ESI-MS m/z calc. 208.0, found; 209.1 (M+1)⁺ ¹H NMR (CDCl₃) δ 7.37-7.18(m, 4H), 6.40 (s, 1H), 4.66 (bs, 2H), 3.97 (s, 2H).

5-(3-Chloro-benzyl)-oxazol-2-ylamine5-(3-Chloro-benzyl)-oxazol-2-ylamine was prepared in a manner analogousto that of the preparation of 5-(2-methoxy-benzyl)-oxazol-2-ylamine toyield the product as a yellow solid (1.2 g, 2.9% from 3-chloroaniline).ESI-MS m/z calc. 208.0, found; 209.2 ¹H NMR(CDCl₃) δ 7.26-7.22 (m, 3H),7.10 (d, J=6.0 Hz, 1H), 6.44 (s, 1H), 4.73 (bs, 2H), 3.82 (s, 2H).

5-(4-Chloro-benzyl)-oxazol-2-ylamine5-(4-Chloro-benzyl)-oxazol-2-ylamine was prepared in a manner analogousto that of the preparation of 5-(2-methoxy-benzyl)-oxazol-2-ylamine toyield the product as a yellow solid (1.6 g, yield=3.86% from4-chloroaniline). ESI-MS m/z calc. 208.0, found; 209.1 ¹H NMR(CDCl₃) δ7.27 (d, J=8.4 Hz, 2H), 7.17 (d, J=8.0 Hz, 2H), 6.38 (s, 1H), 4.66 (bs,2H), 3.81(s, 2H).

Example 3

2-Bromo-3-phenylpropionaldehyde A solution of bromine (15.2 g, 95.1mmol)in 30 mL of dichloromethane was added to a solution of3-phenyl-propionaldehyde (13.4 g, 100 mmol) in dichloromethane (150 mL)at 0° C. over 20 minutes. The reaction mixture was allowed to stir for 2hours and then a saturated aqueous solution of sodium bicarbonate (100mL) was added to the mixture. The organic layer was separated and theaqueous layer was washed with dichloromethane (50 mL). The combinedorganic layers were washed with water, a saturated aqueous solution ofsodium chloride, and then evaporated to dryness to give an orange oil(14.2 g), which was used directly in the next step.

5-Benzyl-oxazol-2-ylamine A mixture of2-bromo-3-phenylpropionaldehyde(14.2 g, crude from above) and urea (7.2g, 0.12 mol) were heated to reflux for 15 hours in 200 mL of ethanol.The solvent was evaporated to dryness and the residue was diluted withdichloromethane (250 mL) and then washed with sodium hydroxide (10%aqueous solution, 100 mL) and water (50 mL). The organic layer wasextracted three times with hydrochloric acid (5% aqueous solution, 250mL). The combined aqueous layers were brought to pH 9 to 10 with a 10%aqeous solution of sodium hydroxide and then extracted three times withdichloromethane (300 mL). The organic layer was dried over sodiumsulfate, evaporated to dryness, and purified by silica gel columnchromatography to give a pale yellow solid. (1.6 g, 9.2 mmol, 9.2% from3-phenyl-propionaldehyde). ESI-MS m/z calc. 174.1, found; 175.1 ¹HNMR(CDCl₃) δ 7.32-7.22 (m, 5H), 6.39 (s, 1H), 4.72 (bs, 2H), 3.84 (s,2H).

Example 4

2-Bromo-3-phenylpropionaldehyde A solution of bromine (15.2 g, 95.1mmol)in 30 mL of dichloromethane was added to a solution of3-phenyl-propionaldehyde (13.4 g, 100 mmol) in dichloromethane (150 mL)at 0° C. over 20 minutes. The reaction mixture was allowed to stir for 2hours and then a saturated aqueous solution of sodium bicarbonate (100mL) was added to the mixture. The organic layer was separated and theaqueous layer was washed with dichloromethane (50 mL). The combinedorganic layers were washed with water, a saturated aqueous solution ofsodium chloride, and then evaporated to dryness to give an orange oil(14.2 g), which was used directly in the next step.

5-benzyl-thioxazol-2-ylamine A mixture of2-bromo-3-phenylpropionaldehyde(14.2 g, crude from above) and urea (7.2g, 0.12 mol) were heated to reflux for 15 hours in 200 mL of ethanol.The solvent was evaporated to dryness and the residue was diluted withdichloromethane (250 mL) and then washed with sodium hydroxide (10%aqueous solution, 100 mL) and water (50 mL). The organic layer wasextracted three times with hydrochloric acid (5% aqueous solution, 250mL). The combined aqueous layers were brought to pH 9 to 10 with a 10%aqeous solution of sodium hydroxide and then extracted three times withdichloromethane (300 mL). The organic layer was dried over sodiumsulfate, evaporated to dryness, and purified by silica gel columnchromatography to give a pale yellow solid. (5.2 g, 27 mmol, 27% from3-phenyl-propionaldehyde). ESI-MS m/z calc. 190.1, found; 191.2 ¹HNMR(CDCl₃) δ 7.32-7.21 (m, 5H), 6.79 (s, 1H), 4.91 (bs, 2H), 3.95 (s,2H).

Example 5

5-(2-Chloro-benzyl)-[1,3,4]oxadiazol-2-ylamine Semicarbazide (9.0 g, 120mmol) was slowly added to a solution of (2-chlorophenyl)acetic acid (10.g, 60 mmol) in phosphorus oxychloride (50 mL). The mixture was stirredat room temperature for 16 hours and then poured into crushed ice (500g). A viscous solid was decanted from the aqueous layer and then theaqueous layer was adjusted to pH 4 to 5 with sodium hydroxide (50%aqueous solution). The resulting precipitate was filtered and thenwashed with sodium carbonate (10% aqueous solution, 100 mL) to give theproduct as a white solid. (5.9 g, 28 mmol, 47%). ESI-MS m/z calc. 209.0,found; 210.1 (M+1)⁺ ¹H NMR(DMSO) δ 7.44-7.30 (m, 4H), 6.88 (s, 2H), 4.13(s, 2H).

Example 6

5-(2-Chloro-benzyl)-[1,3,4]thiadiazol-2-ylamine. A solution ofphosphorous oxychloride (16.6 g, 108 mmol) in 1,4-dioxane (50 mL) wasadded over a period of 30 minutes to a suspension of(2-chlorophenyl)acetic acid (25.0 g, 147 mmol) and thiosemicarbazide(13.3 g, 147 mmol) in 1,4-dioxane (350 mL) at 90° C. The mixture wasstirred at 90° C. for an additional 1.5 hours and then the solvent wasevaporated to dryness. The residue was treated with water (300 mL) andthen made strongly basic with sodium hydroxide (30% aqueous solution).The solid was filtered and then washed with ethyl acetate. The ethylacetate layer was evaporated to give a mixture of5-(2-chloro-benzyl)-[1,3,4]thiadiazol-2-ylamine andN-[5-(2-chloro-benzyl)-[1,3,4]thiadiazol-2-yl]-2-(2-chloro-phenyl)-acetamideas a pale-yellow solid (13.2 g). The solid was refluxed in a mixture ofhydrochloric acid (20% aqueous solution, 250 mL) and ethanol (50 mL) for15 hours and then cooled to room temperature. The resulting precipitatewas collected and then washed with sodium carbonate (10% aqueoussolution, 50 mL) to give 5-(2-chloro-benzyl)-[1,3,4]thiadiazol-2-ylamineas a white solid (5.2 g, 23 mmol, 16%). ESI-MS m/z calc. 225.0, found;226.1 (M+1)⁺ ¹H NMR(DMSO) δ 7.48-7.32 (m, 4H), 4.29 (s, 2H).

Example 7

2-Chloro-3-(2-chloro-phenyl)-propionaldehyde. A solution of sodiumnitrite (15 g, 0.22 mol) in water (40 mL) was slowly added to a solutionof 2-chloroaniline (25.5 g, 0.200 mol) in hydrochloric acid (20% aqueoussolution, 100 mL) at 0 to 5° C. The mixture was stirred for ten minutesand then poured into a cooled solution of acrolein (30. g, 0.56 mol) inacetone (200 mL) containing calcium oxide (4.0 g, 72 mmol), followed bya solution of cuprous chloride (2.0 g, 20 mmol) in acetone (20 mL)containing hydrochloric acid (20% aqueous solution, 4 mL). The mixturewas stirred for 3 hours at room temperature, and then extracted threetimes with dichloromethane (150 mL). The combined organic layers werewashed with a solution of saturated aqueous sodium chloride, dried oversodium sulfate, filtered, and evaporated to dryness to give a blackviscous oil that was used directly in the next procedure.

[2-(2-Chloro-phenyl)-1-methyliminomethyl-ethyl]-methyl-amine. A solutionof methylamine in methanol (27%, 69 g) was slowly added to a solution of2-chloro-3-(2-chloro-phenyl)-propionaldehyde in dichloromethane (20 mL).The reaction mixture was allowed to stir for 12 hours and then usedimmediately in the next procedure.

5-(2-Chloro-benzyl)-1-methyl-1H-imidazol-2-ylamine. A solution ofcyanamide in water (50%, 150 mL) was added to a boiling solution of[2-(2-chloro-phenyl)-1-methyliminomethyl-ethyl]-methyl-amine in methanoland dichloromethane. The pH was brought to 4.5 by continual addition ofan aqueous solution of sulfuric acid (9 M). The mixture was refluxed for2 hours, allowed to cool to room temperature, and adjusted to pH 9through the addition of powdered sodium bicarbonate. The mixture wasextracted three times with dichloromethane (200 mL) and the combinedorganic layers were extracted three times with hydrochloric acid (20%aqueous solution, 150 mL). The aqueous solution was adjusted to pH 10with sodium hydroxide (10% aqueous solution) and extracted three timeswith dichloromethane (150 mL). The combined organic layers were washedwith a saturated aqueous solution of sodium chloride, dried over sodiumsulfate, filtered, and evaporated to dryness to give a black solid whichwas purified by column chromatography to yield the product (5.0 g, 0.23mmol, 11% from 2-chloroaniline) as a brown solid. ESI-MS m/z calc.221.1, found; 222.3 (M+1)⁺ ¹H NMR (CDCl₃): δ 7.30-7.37 (m, 1 H),7.15-7.18 (m, 2 H), 7.03-7.06 (m, 1 H), 6.43 (s, 1 H), 3.94 (s, 2H),3.80 (br, 2H), 3.15 (s, 3 H).

Example 8

3-(2-Chloro-6-fluoro-phenyl)-acrylic acid ethyl ester A solution of(diethoxyphosphoryl)-acetic acid ethyl ester (87 g, 0.39 mol) intetrahydrofuran (100 mL) was slowly added to a suspension of sodiumhydride (60% in mineral oil, 15 g, 0.39 mol) in tetrahydrofuran (200 mL)at 0° C. After stirring for 20 minutes, a solution of2-chloro-6-fluoro-benzaldehyde (40 g, 0.26 mol) in tetrahydrofuran (100mL) while maintaining the temperature at 0° C. The mixture was heated to50° C. for 1 hour and then cooled to room temperature. The reaction wasquenched by addition of a saturated aqueous solution of ammoniumchloride (300 mL). The layers were separated and the aqueous layer wasextracted with ether. The combined organic layers were washed with asaturated aqueous solution of sodium chloride, dried over sodiumsulfate, and evaporated to dryness to give3-(2-chloro-6-fluoro-phenyl)-acrylic acid ethyl ester, which was useddirectly in the next step. ¹H NMR (CDCl₃) δ: 7.89 (d, 1 H, J=16.4 Hz),7.26-7.06 (m, 2 H), 7.06-7.02 (m, 1 H), 6.72 (d, 1 H, J=16.4 Hz), 4.28(q, 2 H, J=7.6 Hz), 1.34 (t, 3 H, J=7.6 Hz).

3-(2-chloro-6-fluoro-phenyl)-propan-1-ol A solution of3-(2-chloro-6-fluoro-phenyl)-acrylic acid ethyl ester in driedtetrahydrofuran (300 mL) was added to a suspension of lithium aluminumhydride (30 g, 0.78 mol) in anhydrous tetrahydrofuran (200 mL) at 0° C.The reaction mixture was stirred for 3 hours, and then cooled to 0° C.and quenched by the addition of water (30 g) and a 10% aqueous solutionof sodium hydroxide (30 mL). The resulting solid was filtered, washedwith tetrahydrofuran, and then purified by silica gel columnchromatography to afford 3-(2-chloro-6-fluoro-phenyl)-propan-1-ol (21 g,0.11 mol, 43% from 2-chloro-6-fluoro-benzaldehyde).

3-(2-Chloro-6-fluoro-phenyl)-propionaldehyde A solution of pyridinecomplex with sulfur trioxide (40.35 g, 0.225 mol) in dimethylsulfoxide(50 mL) was slowly added to a solution of3-(2-chloro-6-fluoro-phenyl)-propan-1-ol (20.6 g, 0.109 mol) andtriethylamine (25.8 g, 0.225 mol) in dichloromethane (250 mL) at 0° C.The reaction was allowed to stir for 30 minutes, and then it was pouredinto a saturated aqueous solution of sodium chloride and extracted withethyl acetate. The combined organic layers were washed twice with waterand then with a saturated aqueous solution of sodium chloride, driedover magnesium sulfate and evaporated to dryness. The crude product waspurified by silica gel chromatography to afford3-(2-Chloro-6-fluoro-phenyl)-propionaldehyde (15 g, 80 mmol, 74%), ¹HNMR (CDCl₃): δ 9.83 (s, 1 H), 7.24-7.00 (m, 2 H), 6.99-6.94 (m, 1 H),3.13-3.09 (m, 2 H), 2.75-2.71 (m, 2 H).

2-Bromo-3-(2-chloro-6-fluoro-phenyl)-propionaldehyde A solution ofbromine (11 g, 0.081 mol) in dichloromethane (50 mL) was slowly added toa solution of 3-(2-chloro-6-fluoro-phenyl)-propionaldehyde (15 g, 0.081mol) in dichloromethane (250 mL) at 0° C. The mixture was stirredovernight at room temperature and then the solvent was removed to givethe crude product which was used directly in the next step.

5-(2-Chloro-6-fluoro-benzyl)-thiazol-2-ylamine. A mixture of2-bromo-3-(2-chloro-6-fluoro-phenyl)-propionaldehyde (crude from laststep) and thiourea (6.4 g, 0.084 mol) in ethanol (250 mL) was refluxedovernight. The reaction mixture was evaporated to dryness anddichloromethane (50 mL) was added to the residue. The mixture wasacidified with concentrated hydrochloric acid to between pH 2 and 3. Theprecipitated solid was filtered, and washed with dichloromethane to givethe hydrochloride salt (7.7 g, 34.1%). ESI-MS m/z calc. 242.0, found;243.2 (M+1)⁺ ¹H NMR (CDCl₃): δ 8.62 (s, 2 H), 7.37-7.36 (m, 2 H),7.29-7.26 (m, 1 H), 6.98 (s, 1 H), 4.05 (s, 2 H).

Example 9

(2-Methoxy-phenyl)-acetic acid methyl ester A solution of methyl iodide(188 g, 1.33 mole) in acetonitrile (200 mL) was slowly added to amixture of (2-hydroxy-phenyl)-acetic acid (80 g, 0.53 mol) and potassiumcarbonate (254 g, 1.84 mol) in acetonitrile (800 mL) under reflux. Thereaction was heated to reflux for 15 hours. The reaction mixture wascooled and the precipitate was removed by filtration. The filtrate wasevaporated to dryness to give the crude product (90. g, 0.50 mol, 94%).

2-(2-Methoxy-phenyl)-ethanol Lithium aluminum hydride (21 g, 0.55 mol)was added to a solution of (2-methoxy-phenyl)-acetic acid methyl ester(90 g, 0.50 mol) in anhydrous tetrahydrofuran (500 mL) at 0° C. Afterstirred at 0° C. for 30 minutes, the mixture was treated with sodiumhydroxide (5% aqueous solution, 180 g). The mixture was extracted threetimes with ethyl acetate (400 mL) and the combined organic layers werewashed with a saturated aqueous solution of sodium chloride, dried oversodium sulfate, filtered, and evaporated to dryness to give2-(2-methoxy-phenyl)-ethanol (43 g, 0.28 mol, 57%), which was useddirectly in the next step.

(2-Methoxy-phenyl)-acetaldehyde A solution of pyridine complex withsulfur trioxide (134 g, 0.842 mol) in dimethylsulfoxide (150 mL) wasslowly added to a solution of 2-(2-methoxy-phenyl)-ethanol (43 g, 0.28mol) and triethylamine (86 g, 0.85 mol) in dichloromethane (500 mL) at0° C. After being stirred 30 min, the mixture was poured into asaturated aqueous solution of sodium chloride and the organic layer waswashed with dilute hydrochloric acid, a saturated aqueous solution ofsodium chloride, dried over sodium sulfate and evaporated to dryness togive (2-methoxy-phenyl)-acetaldehyde (36 g, 0.24 mol, 86%) which wasused in the next step directly.

{5-[1-Hydroxy-2-(2-methoxy-phenyl)-ethyl]-thiazol-2-yl}-carbamic acidtert-butyl ester A solution of n-butyl lithium (2.5 M, 250 mL, 0.62 mol)was added to a solution of N-Boc-2-aminothiazole (56 g, 0.28 mol) inanhydrous tetrahydrofuran (500 mL) was added at −78° C. After theaddition was completed, the mixture was allowed to stir at −78° C. forone hour. A solution of (2-methoxy-phenyl)-acetaldehyde (36 g, 0.24 mol)in tetrahydrofuran (100 mL) was slowly added to the reaction mixturewhile maintaining a temperature of −78° C. The mixture was allowed towarm to room temperature and stirred for 15 hours. The reaction wasquenched with a saturated aqueous solution of ammonium chloride (1000mL) and extracted three times with ethyl acetate (400 mL). The combinedorganic layers were washed with a saturated aqueous solution of sodiumchloride, dried over sodium sulfate, and evaporated to dryness. Thecrude product was purified by column chromatography to give the pureproduct (28 g, 0.080 mol, 28%).

5-[2-(2-Methoxy-phenyl)-ethyl]-thiazol-2-ylamine A mixture of{5-[1-Hydroxy-2-(2-methoxy-phenyl)-ethyl]-thiazol-2-yl}-carbamic acidtert-butyl ester (28 g, 0.080 mol), triethylsilane (130 g, 1.12 mol) andtrifluoroacetic acid (250 g, 2.24 mol) in dichloromethane (500 mL) wasstirred at room temperature for 15 hours. The mixture was evaporated todryness and then stirred in water. The solid was filtered and washedwith ether to give 5-[2-(2-Methoxy-phenyl)-ethyl]-thiazol-2-ylamine as atrifluoracetic acid salt (11 g, 0.033 mol, yield 42%). ESI-MS m/z cacl.234.08, found 235.16 (M+1)⁺ ¹H NMR(CDCl₃) δ 8.81 (br, 2H), 7.20 (t, 1H),7.12 (d, 1H), 6.95 (m, 2H), 6.85 (t, 1H), 3.76 (s, 3H), 2.83 (d, 2H),2.80 (d, 2H).

Example 10

2-Bromo-3-oxo-hexanoic acid ethyl ester 3-Oxo-hexanoic acid ethyl ester(4.0 mL, 25 mmol) and magnesium perchlorate (1.7 g, 7.6 mmol) was placedin 500 mL of ethyl acetate and allowed to stir for 5 minutes.N-Bromosuccinimide (4.7 g, 26 mmol) was added and the reaction mixturewas allowed to stir for 15 minutes, at which time thin-layerchromatography (10% ethyl acetate in hexanes, SiO₂, 254 nm irradiation)indicated the reaction was complete. The reaction mixture was dilutedwith 500 mL of ethyl ether and washed three times with an equal volumeof saturated aqueous sodium chloride. The organic layer was then driedover sodium sulfate and evaporated to dryness. This material was used inthe next step without further purification.

2-Amino-4-propyl-thiazole-5-carboxylic acid ethyl ester2-Bromo-3-oxo-hexanoic acid ethyl ester (5.9 g, 25 mmol), was dissolvedin 60 mL of ethanol containing triethylamine (4.2 mL, 30 mmol) andthiourea (1.9 g, 25 mmol). The colorless solution was protected fromlight and allowed to stir for 16 hours. The resulting red suspension wasevaporated to dryness and dissolved in a minimum of dichloromethane.This solution was washed three times with an equal volume of a saturatedaqueous solution of sodium bicarbonate, followed by a saturated aqueoussolution of sodium chloride. The organic layer was separated andfiltered to remove a fine red precipitate which remained suspended inthe organic phase. The solvent was removed and then the solid wasdissolved in a minimum of 50/50 (v/v) ethyl acetate and 1 N aqueoussolution of hydrochloric acid. The layers were separated and the aqueouslayer was washed with an equal volume of ethyl acetate. After discardingthe organic layers, the aqueous layer was then placed in an ice bathwith an equal volume of ethyl acetate. Sodium hydroxide (1N) was thenslowly added with vigorous swirling until the aqueous phase was basic.The layers were separated and the aqueous layer was washed twoadditional times with ethyl acetate. The combined organic layers werewashed three times with an equal volume of a solution of saturatedaqueous sodium bicarbonate followed by a solution of saturated aqueoussodium chloride. The organic layer was then dried over sodium sulfateand evaporated to dryness to yield a pale yellow solid (1.8 g, 8.4 mmol,34%) ESI-MS m/z calc. 214.1, found; 215.3 (M+1)⁺ Retention time 1.90minutes.

Example 11

2-Amino-thiazole-4-carboxylic acid ethyl ester A mixture of ethylbromopyruvate (100 g, 80% purity, 0.41 mol), thiourea (31 g, 0.41 mol)and ethanol (500 mL) was heated to reflux for 12 hours. The solvent wasevaporated to dryness and the residue was washed with ether. The solidwas suspended in a saturated aqueous solution of sodium bicarbonate (500mL) for 30 minutes. The solid was filtered, washed with water, and driedover sodium sulfate to give 2-amino-thiazole-4-carboxylic acid ethylester (45 g, 0.26 mol, 63%) as an off-white solid.

2-tert-butoxycarbonylamino-thiazole-4-carboxylic acid ethyl esterDi-tert-butyl dicarbonate (57 g, 0.26 mol) in dichloromethane (100 mL)was slowly added to a solution of 2-amino-thiazole-4-carboxylic acidethyl ester (45 g, 0.26 mole) and dimethyl-pyridin-4-yl-amine (1 g,0.008 mol) in dichloromethane (500 mL) at 0° C. The mixture was stirredat room temperature overnight and then diluted with water. The organiclayer was washed with a saturated aqueous solution of sodium chloride,dried over sodium sulfate, filtered, and evaporated to dryness to give acrude product. The product was purified by column chromatography toafford the pure product (43 g, 0.16 mol, 62%) as a white solid.

2-tert-butoxycarbonylamino-5-[hydroxy-(2-methoxy-phenyl)-methyl]-thiazole-4-carboxylicacid ethyl ester Butyl lithium (2.5 M, 53 mL, 132 mmol) was slowly addedto a solution of 2-tert-butoxycarbonylamino-thiazole-4-carboxylic acidethyl ester (16.4 g, 60 mmol) in anhydrous tetrahydrofuran (250 mL) at−78° C. under a nitrogen atmosphere. A solution of 2-methoxybenzaldehyde(10.89 g, 79.99 mmol) in tetrahydrofuran (50 mL) was then added dropwiseat −78° C. The mixture was warmed slowly to room temperature and stirredfor 15 hours. The reaction mixture was quenched with a saturated aqueoussolution of ammonium chloride (500 mL). The separated aqueous layer wasextracted with ethyl acetate and the combined organic layers were washedwith a saturated aqueous solution of sodium chloride, dried over sodiumsulfate, filtered, and evaporated to dryness. The crude product was thenpurified by column chromatography to yield the pure product (12 g, 29mmol, 48%).

2-Amino-5-(2-methoxy-benzyl)-thiazole-4-carboxylic acid ethyl ester Amixture of2-tert-butoxycarbonylamino-5-[hydroxy-(2-methoxy-phenyl)-methyl]-thiazole-4-carboxylicacid ethyl ester (12 g, 29 mmol), triethylsilane (52 g, 45 mmol), andtrifluoroacetic acid (100. g, 89.6 mmol) was stirred for 15 hours indichloromethane (150 mL). The mixture was evaporated to dryness and thendiluted with water. The precipitated solid was filtered and washed withwater, diethyl ether, and petroleum ether to give the pure targetmolecule as a trifluoroacetic acid salt (7.6 g, 19 mmol, 64%). ESI-MSm/z calc. 292.09, found; 292.25 (M+1)⁺ ¹H NMR(CDCl₃) δ 7.7 (br, 2H),7.24 (t, 1H), 7.17 (d, 1H), 6.99 (d, 1H), 6.88 (t, 1H), 4.22-4.25 (m,4H), 3.78 (s, 3H), 1.26 (t, 3H).

Example 12

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid{5-[(2-chloro-phenyl)-hydroxy-methyl]-thiazol-2-yl}-amide1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-(2-chloro-benzoyl)-thiazol-2-yl]-amide (1.0 g, 2.3 mmol) wassuspended in 150 mL of anhydrous methanol. Sodium borohydride (1.3 g, 35mmol) was slowly added and the resulting pale yellow solution wasallowed to stir for 1 hour at room temperature. The crude product wasevaporated to dryness and then dissolved in a minimum of ethyl acetate.The organic was washed three times with an equal volume of 1Nhydrochloric acid, saturated aqueous sodium bicarbonate, and saturatedaqueous sodium chloride. The organic layer was then dried over sodiumsulfate, filtered, and evaporated to dryness to yield the product as abeige solid (0.64 g, 1.5 mmol, 63%) ESI-MS m/z calc. 428.1, found; 429.5(M+1)⁺ Retention time 3.17 minutes.

Example 13

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid(5-piperidin-1-yl-thiazol-2-yl)-amide1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid(5-bromo-thiazol-2-yl)-amide (110.4 mg, 0.3125 mmol) was dissolved in 3mL of N,N-dimethylformamide containing piperidine (148.2 μL, 1.500mmol). The reaction mixture was placed in a sealed tube and subjected tomicrowave irradiation for 5 minutes at 100° C. The resulting crudeproduct was purified by reverse-phase preparative liquid chromatographyto give the pure product (3.44 mg, 0.00926 mmol, 2.96%) ESI-MS m/z calc.371.1, found; 372.3 (M+1)⁺ Retention time 3.29 minutes.

Example 14

General Procedure: The appropriate alcohol (2 equivalents) was dissolvedin a solution of 10% N,N-dimethylformamide (DMF) in tetrahydrofuran(THF). Sodium hydride (2.1 equivalents) was added and the reaction wasstirred for 5 minutes under an atmosphere of nitrogen. A solution of theappropriate halide (1 equivalent)in a solution of 10% DMF THF was slowlyadded to the reaction mixture. The mixture was allowed to stir for 10minutes and then ether was added. The reaction mixture was washed with1N sodium hydroxide. The organic layer was dried over sodium sulfate,filtered, and evaporated to dryness. The crude product was purified onsilica gel to yield the pure product.

Specific Example

5-(2-Chloro-phenoxy)-thiazol-2-ylamine 2-Chloro-phenol (1.7 mL, 16.0mmol) was dissolved in a mixture of 36 mL of tetrhydrofuran (THF) and 4mL of N,N-dimethylformamide (DMF). Sodium hydride (60% dispersion inoil, 0.700 g, 17.5 mmol) was added and the reaction was stirred for 5minutes under an atmosphere of nitrogen. A solution of5-bromo-thiazol-2-ylamine hydrobromide (2.1 g, 8.0 mmol)in 18 mL of THFand 2 mL of DMF was slowly added to the reaction mixture. The mixturewas allowed to stir for 10 minutes and then 100 mL of ether was added.The reaction mixture was washed with 1N sodium hydroxide. The organiclayer was dried over sodium sulfate, filtered, and evaporated todryness. The crude product was purified on silica gel using a gradientof 50-99% ethyl acetate in hexanes to yield the product as a brown solid(0.3546 g, 1.564 mmol, 19%)

Example 15

1-Cyclopropylethynyl-4-methoxy-benzene Cyclopropylethyne (15 g, 0.22mol) was slowly added to a mixture of 4-iodoanisole (30. g, 0.13 mol),copper (I) iodide (1.2 g, 0.0063 mol), and triethylamine (30. g, 0.30mol) in tetrahydrofuran (THF, 400 mL). After being stirred for 4 hours,the resulting precipitate was filtered and washed with THF. The combinedfiltrate was evaporated to dryness and the residue was purified bycolumn chromatography to afford 1-cyclopropylethynyl-4-methoxy-benzeneas a yellow oil (15 g, 0.087 mol, 67%). ¹H NMR (CDCl₃): δ 7.31 (d, J=8.8Hz, 2 H), 6.80 (d, J=8.8 Hz, 2 H), 3.79 (s, 3 H), 1.43 (m, 1 H), 0.85(m, 2 H), 0.78 (m, 2 H).

1-(4-Methoxy-phenylethynyl)-cyclopropanecarboxylic acid and2-Cyclopropylethynyl-5-methoxy-benzoic acid. To a solution of1-cyclopropylethynyl-4-methoxy-benzene (7.5 g, 0.043 mol) in THF (250mL) was added dropwise n-butyl lithium (n-BuLi, 21 mL, 0.052 mol) at−78° C. under nitrogen. After being stirring for 15 minutes, thesolution was warmed to room temperature and stirred for another hour.The solution was cooled to −78° C. and gaseous carbon dioxide wasintroduced to the solution for 1 hour. The mixture was warmed to −20° C.and quenched with water (100 mL). The solution was washed with ether andthe separated aqueous layer was acidified to pH 4. The mixture wasextracted with ether and the combined organic layer was washed with asaturated aqueous solution of sodium chloride, dried with sodiumsulfate, and evaporated to dryness to afford a mixture of1-(4-methoxy-phenylethynyl)-cyclopropanecarboxylic acid and2-cyclopropylethynyl-5-methoxy-benzoic acid as a yellow solid (5.0 g,0.023 mmol).

1-(4-Methoxy-phenylethynyl)-cyclopropanecarboxylic acid methyl ester and2-Cyclopropylethynyl-5-methoxybenzoic acid methyl ester. Iodomethane(3.6 g, 0.026 mol) was slowly added to a mixture of1-(4-methoxy-phenylethynyl)-cyclopropanecarboxylic acid and2-cyclopropylethynyl-5-methoxy-benzoic acid (5.0 g) and potassiumcarbonate (4.8 g, 0.034 mol) in DMF (100 mL). After stirring for 4hours, the mixture was diluted with ether (200 mL) and washed withwater. The ether layer was dried over sodium sulfate, evaporated todryness, and the residue was purified by column chromatography to yield1-(4-methoxy-phenylethynyl)-cyclopropanecarboxylic acid methyl ester asa yellow oil (2.2 g). ¹H NMR (CDCl₃): δ 7.36 (d, J=8.8 Hz, 2 H), 6.81(d, J=8.8 Hz. 2 H), 3.79 (s, 3 H), 3.76 (s, 3 H), 1.62 (m, 2 H), 1.41(m, 2 H). 2-cyclopropylethynyl-5-methoxy-benzoic acid methyl ester (1.0g). ¹H NMR(CDCl₃): δ 7.89 (d, J=2.4 Hz, 1 H), 7.46 (dd, J₁=8.4 Hz,J₂=2.4 Hz, 1 H), 6.8 (d, J=8.4 Hz, 1 H), 3.88 (s, 3 H), 3.87 (s, 3 H),1.43 (m, 1 H), 0.86 (m, 2 H), 0.78 (m, 2 H).

1-(4-Methoxy-phenylethynyl)-cyclopropanecarboxylic acid A mixture of1-(4-methoxy-phenylethynyl)-cyclopropanecarboxylic acid methyl ester(2.2 g, 9.4 mmol), lithium hydroxide (0.6 g, 14 mmol) in THF (30 mL),and water (30 mL) was stirred for 6 hours. The solution was washed withether and the aqueous layer was acidified to pH=4 and then extractedwith ether. The organic layer was dried over sodium sulfate, andevaporated to dryness to yield1-(4-methoxy-phenylethynyl)-cyclopropanecarboxylic acid as a white solid(2.0 g, 0.0092 mmol, 98%). ESI-MS m/z calc. 216.1, found; 217.16 (M+1)⁺¹H NMR(CDCl₃): δ 7.35 (d, J=8.8 Hz, 2 H), 6.80 (d, J=8.8 Hz. 2 H), 3.79(s, 3 H), 1.69 (m, 2 H), 1.47 (m, 2 H).

Example 16

1-(4-methoxy-benzyl)-cyclopropanecarbonitrile Sodiumbis(trimethylsilyl)amide (2 M in tetrahydrofuran (THF), 37.5 mL, 75.0mmol) was slowly added to a solution of cyclopropanecarbonitrile (3.35g, 49.9 mmol) in THF (30 mL) at room temperature. The reaction mixturewas stirred for 20 minutes and then a solution1-chloromethyl-4-methoxy-benzene (7.83 g, 50.0 mmol) in THF was added.The mixture was heated to reflux for 3 hours and then quenched with asaturated aqueous solution of ammonium chloride (50 mL). The separatedaqueous layer was extracted three times with ethyl acetate (50 mL). Thecombined organic layer was washed with a saturated aqueous solution ofsodium bicarbonate and a saturated aqueous solution of sodium chloride,dried over sodium sulfate, filtered, and evaporated to dryness to givecrude 1-(4-methoxy-benzyl)-cyclopropanecarbonitrile, which was useddirectly in the next step.

1-(4-methoxy-benzyl)-cyclopropanecarboxylic acid The crude1-(4-methoxybenzyl)-cyclopropanecarbonitrile was refluxed in sodiumhydroxide (10% aqueous solution, 30 mL) overnight. The mixture wasdiluted with water (30 mL) and then washed with ethyl ether. The aqueousphase was acidified with 2 M hydrochloric acid to pH 5 and extractedthree times with ethyl acetate (50 mL). The combined organic layer waswashed with a saturated aqueous solution of sodium chloride, dried oversodium sulfate, filtered, and evaporated to dryness to give the pureproduct. (5.6 g, 54.4% from cyclopropanecarbonitrile). ESI-MS m/z calc.206.1, found; 206.9 (M+1)⁺ ¹H NMR (DMSO): δ: 12.15 (s, 1 H), 7.13 (d,J=8.4, 2 H), 6.80 (d, J=8.4, 2 H), 3.69 (s, 3 H), 2.78 (s, 2 H),1.07-1.04 (m, 2 H), 0.76-0.74 (m, 2 H).

Example 17

2,2-Difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester A solutionof 5-bromo-2,2-difluoro-benzo[1,3]dioxole (11.8 g, 50.0 mmol) andtetrakis(triphenylphosphine)palladium (0) (Pd(PPh₃)₄, 5.78 g, 5.00 mmol)in methanol (20 mL) containing acetonitrile (30 mL) and triethylamine(10 mL) was stirred under a carbon monoxide atmosphere (55 PSI) at 75°C. (oil bath temperature) for 15 hours. The cooled reaction mixture wasfiltered and the filtrate was evaporated to dryness. The residue waspurified by silica gel column chromatography to give crude2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester (11.5 g),which was used directly in the next step.

(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-methanol Crude2,2-Difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester (11.5 g)dissolved in 20 mL of anhydrous tetrahydrofuran (THF, 20 mL) was slowlyadded to a suspension of lithium aluminum hydride (4.10 g, 106 mmol) inanhydrous THF (100 mL) at 0° C. The mixture was then warmed to roomtemperature. After being stirred at room temperature for 1 hour, thereaction mixture was cooled to 0° C. and treated with water (4.1 g),followed by sodium hydroxide (10% aqueous solution, 4.1 mL). Theresulting slurry was filtered and washed with THF. The combined filtratewas evaporated to dryness and the residue was purified by silica gelcolumn chromatography to give(2,2-difluoro-benzo[1,3]dioxol-5-yl)-methanol (7.2 g, 38 mmol, 76% overtwo steps) as a colorless oil.

5-Chloromethyl-2,2-difluoro-benzo[1,3]dioxole Thionyl chloride (45 g, 38mmol) was slowly added to a solution of(2,2-difluoro-benzo[1,3]dioxol-5-yl)-methanol (7.2 g, 38 mmol) indichloromethane (200 mL) at 0° C. The resulting mixture was stirredovernight at room temperature and then evaporated to dryness. Theresidue was partitioned between an aqueous solution of saturated sodiumbicarbonate (100 mL) and dichloromethane (100 mL). The separated aqueouslayer was extracted with dichloromethane (150 mL) and the combinedorganic layer was dried over sodium sulfate, filtrated, and evaporatedto dryness to give crude 5-chloromethyl-2,2-difluoro-benzo[1,3]dioxole(4.4 g) which was used directly in the next step.

(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile A mixture of crude5-chloromethyl-2,2-difluoro-benzo[1,3]dioxole (4.4 g) and sodium cyanide(1.36 g, 27.8 mmol) in dimethylsulfoxide (50 mL) was stirred at roomtemperature overnight. The reaction mixture was poured into ice andextracted with ethyl acetate (300 mL). The combined organic layers weredried over sodium sulfate and evaporated to dryness to give crude(2,2-difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile (3.3 g) which was useddirectly in the next step.

1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile Sodiumhydroxide (50% aqueous solution, 10 mL) was slowly added to a mixture ofcrude (2,2-difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile,benzyltriethylammonium chloride (3.00 g, 15.3 mmol), and1-bromo-2-chloroethane (4.9 g, 38 mmol) at 70° C. The mixture wasstirred overnight at 70° C. and the reaction mixture was diluted withwater (30 mL) and extracted with ethyl acetate. The combined organiclayers were dried over sodium sulfate and evaporated to dryness to givecrude 1-(2,2-difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile,which was used directly in the next step.

1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarboxylic acid The1-(2,2-difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile (crudefrom the last step) was refluxed in 10% aqueous sodium hydroxide (50 mL)for 2.5 hours. The cooled reaction mixture was washed with ether (100mL) and the aqueous phase was acidified to pH 2 with 2M hydrochloricacid. The precipitated solid was filtered to give1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarboxylic acid as awhite solid (0.15 g, 0.62 mmol, 1.6% over four steps). ESI-MS m/z calc.242.04, found 241.58 (M−1)⁻ ¹H NMR (CDCl₃): δ 7.14-7.04 (m, 2 H),6.98-6.96 (m, 1 H), 1.74-1.64 (m, 2 H), 1.26-1.08 (m, 2 H).

Example 18

General procedure: Sodium hydroxide (50% aqueous solution, 7.4equivalents) was slowly added to a mixture of the appropriate phenylacetonitrile, benzyltriethylammonium chloride (1.1 equivalents), and theappropriate dihalo compound (2.3 equivalents) at 70° C. The mixture wasstirred overnight at 70° C. and the reaction mixture was diluted withwater (30 mL) and extracted with ethyl acetate. The combined organiclayers were dried over sodium sulfate and evaporated to dryness to givethe crude cyclopropanecarbonitrile, which was used directly in the nextstep.

The crude cyclopropanecarbonitrile was refluxed in 10% aqueous sodiumhydroxide (7.4 equivalents) for 2.5 hours. The cooled reaction mixturewas washed with ether (100 mL) and the aqueous phase was acidified to pH2 with 2M hydrochloric acid. The precipitated solid was filtered to givethe cyclopropanecarboxylic acid as a white solid.

Specific Example

1-(3,4-Difluoro-phenyl)-cyclopropanecarbonitrile A 50% aqueous solutionof sodium hydroxide (18 g, 46 mmol) was slowly added to a mixture of(3,4-difluoro-phenyl)-acetonitrile (7.1 g, 46 mmol),benzyltriethylammonium chloride (0.26 g, 1.2 mmol) and1-bromo-2-chloroethane (15 g, 105 mmol) at 70° C. The mixture wasstirred overnight at 70° C. The cooled reaction mixture was diluted withwater (50 mL) and extracted three times with ethyl acetate (50 mL). Thecombined organic layers were washed with a saturated aqueous solution ofsodium chloride, dried over sodium sulfate, and evaporated to dryness togive crude 1-(3,4-difluoro-phenyl)-cyclopropanecarbonitrile which wasused directly in the next step. ¹H NMR (CDCl₃): δ 7.18-7.03 (m, 3 H),1.79-1.69 (m, 2 H), 1.43-1.38 (m, 2 H).

1-(3,4-Difluoro-phenyl)-cyclopropanecarboxylic acid. The1-(3,4-Difluoro-phenyl)-cyclopropanecarbonitrile (4.9 g crude from laststep) was refluxed in sodium hydroxide (10% aqueous solution, 100 mL)for 2.5 hours. The reaction mixture was washed twice with ethyl ether(100 mL). The aqueous phase was cooled to 0° C. and brought to betweenpH 2 and 3 with concentrated hydrochloric acid. The aqueous layer wasthen extracted twice with ethyl acetate (100 mL). The combined organiclayers were dried with anhydrous sodium sulfate, filtered, andevaporated to dryness to give a white solid (4.0 g, 20 mmol, 43.5%, twosteps). ESI-MS m/z calc. 198.05, found 197.05 (M−H⁺) ¹H NMR (CDCl₃): δ10.9 (br.s, 1 H), 7.16-7.02 (m, 3 H), 1.66-1.60 (m, 2 H), 1.27-1.21 (m,2 H).

Example 19

General procedure: Benzyltriethylammonium chloride (0.025 equivalents)and the appropriate dihalo compound (2.5 equivalents) was added to asubstituted phenyl acetonitrile. The mixture was heated to 70° C. andthen 50% sodium hydroxide (10 equivalents) was added dropwise. Thereaction was stirred at 70° C. for 12-24 hours to insure completeformation of the cyclopropyl moity and then heated to 150° C. for 24-48hours to insure complete conversion from the nitrile to the carboxylicacid. The dark brown/black reaction mixture was diluted with water andextracted with dichloromethane three times to remove side products. Thebasic aqueous solution was acidified with concentrated hydrochloric acidto pH less than one and the precipitate which began to form at pH 4 wasfiltered and washed with 1 M hydrochloric acid two times. The solidmaterial was dissolved in dichloromethane and extracted two times with 1M hydrochloric acid and one time with a saturated aqueous solution ofsodium chloride. The organic solution was dried over sodium sulfate andevaporated to dryness to give the cyclopropanecarboxylic acid a whitesolid. Yields and purities were typically greater than 90%.

Specific Example

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid. A mixture ofbenzo[1,3]dioxole-5-carbonitrile (5.10 g 31.7 mmol),1-bromo-2-chloro-ethane (9.000 mL 108.6 mmol), andbenzyltriethylammonium chloride (0.181 g 0.795 mmol) was heated to 70°C. and then 50% (wt./wt.) aqueous sodium hydroxide (26 mL) was slowlyadded. The reaction was stirred at 70° C. for 88 hours and then heatedto 130° C. for 24 hours. The dark brown/black reaction mixture wasdiluted with water (400 mL) and extracted twice with equal volumes ethylacetate and dichloromethane. The basic aqueous solution was acidifiedwith concentrated hydrochloric acid to pH less than one and theprecipitate was filtered and washed with 1 M hydrochloric acid. Thesolid material was dissolved in dichloromethane (400 mL) and extractedtwice with equal volumes of 1 M hydrochloric acid and once with asaturated aqueous solution of sodium chloride. The organic solution wasdried over sodium sulfate and evaporated to dryness to give a white toslightly off-white solid (5.23 g, 25.4, 80.1%. ESI-MS m/z calc. 206.06,found 207.1 (M+1)⁺. Retention time of 2.37 minutes. ¹H NMR (400 MHz,DMSO-d₆) δ 1.07-1.11 (m, 2H), 1.38-1.42 (m, 2H), 5.98 (s, 2H), 6.79 (m,2H), 6.88 (m, 1H), 12.26 (s, 1H).

Example 20

General Procedure: One equivalent of the appropriate carboxylic acid andone equivalent of the appropriate amine were dissolved inN,N-dimethylformamide (DMF) containing triethylamine (3 equivalents).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) was added and the solution was allowed tostir. The crude product was purified by reverse-phase preparative liquidchromatography to yield the pure product.

Specific Examples

1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid benzothiazol-2-ylamideBenzothiazol-2-ylamine (30. mg, 0.20 mmol) and1-(4-methoxy-phenyl)-cyclopropanecarboxylic acid (38 mg, 0.20 mmol) weredissolved in N,N-dimethylformamide (DMF, 1 mL) containing triethylamine(84 μL, 0.60 mmol).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 80 mg, 0.21 mmol) was added and the solutionwas allowed to stir at room temperature for 16 hours. The crude productwas purified by reverse-phase preparative liquid chromatography to yieldthe pure product (17 mg, 0.052 mmol, 26%). ESI-MS m/z calc. 324.1, found325.0 (M+1)⁺. Retention time of 3.48 minutes.

Example 21

1-(4-Methoxy-phenyl)-cyclopentanecarboxylic acid[5-(2-chloro-benzyl)-thiazol-2-yl]-amide.5-(2-Chloro-benzyl)-thiazol-2-ylamine (45 mg, 0.20 mmol) and1-(4-Methoxy-phenyl)-cyclopentanecarboxylic acid (44 mg, 0.20 mmol) weredissolved in N,N-dimethylformamide (2 mL) containing triethylamine (84.1□L, 0.600 mmol). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 84 mg, 0.22 mmol) was added and the solutionwas allowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography. ESI-MS m/z calc. 426.1,found; 427.2 (M+1)⁺ Retention time 3.97 minutes. ¹H NMR (400 MHz, CD₃OD)d 1.62-1.84 (m, 4H), 1.95-2.17 (m, 2H), 2.41-2.62 (m, 2H), 3.78 (s, 3H),4.22 (s, 2H), 6.90 (d, J=8.8 Hz, 2H), 7.03-7.49 (m, 7H).

Example 22

General Procedure: One equivalent of the appropriate carboxylic acid andone equivalent of the appropriate amine were dissolved in acetonitrilecontaining triethylamine (3 equivalents).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) was added and the solution was allowed tostir. The crude product was purified by reverse-phase preparative liquidchromatography to yield the pure product.

Specific Examples

2-[(1-Benzo[1,3]dioxol-5-yl-cyclopropanecarbonyl)-amino]-4-propyl-thiazole-5-carboxylicacid ethyl ester 2-Amino-4-propyl-thiazole-5-carboxylic acid ethyl ester(0.50 g, 2.3 mmol) and 1-benzo[1,3]dioxol-5-yl-cyclopropanecarboxylicacid (0.48 g, 2.3 mmol) were dissolved in acetonitrile (15 mL)containing triethylamine (0.66 mL, 4.7 mmol).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 0.89 g, 2.3 mmol) was added and the solutionwas allowed to stir at 65° C. for four hours. The crude product wasevaporated to dryness and purified by column chromatography on silicagel using a gradient of 10-99% ethyl acetate in hexanes. The purefractions were combined and evaporated to dryness to yield a white solid(0.58 g, 1.4 mmol, 61%). ESI-MS m/z calc. 402.1, found 403.3 (M+1)⁺.Retention time of 3.78 minutes. ¹H NMR (400 MHz, CD₃CN) δ 0.84 (t, J=7.4Hz, 3H), 1.25-1.35 (m, 5H), 1.50-1.68 (m, 4H), 2.91 (t, J=7.5 Hz, 2H),4.28 (q, J=7.1 Hz, 2H), 6.00 (s, 2H), 6.81 (d, J=7.7 Hz, 1H), 6.95-6.98(m, 2H), 9.22 (s, 1H).

Example 23

1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-chloro-benzyl)-thiazol-2-yl]-amide5-(2-Chloro-benzyl)-thiazol-2-ylamine (0.250 g, 1.11 mmol) and1-(4-methoxy-phenyl)-cyclopropanecarboxylic acid (0.213 g, 1.11 mmol)were dissolved in acetonitrile (20 mL) containing triethylamine (0.28mL, 2.0 mmol). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 0.494 g, 1.3 mmol) was added and the solutionwas allowed to stir for 16 hours. The crude product was evaporated todryness and purified by column chromatography on silica gel using agradient of 5-40% ethyl acetate in hexanes. The pure fractions werecombined and evaporated to dryness to yield a white solid (0.2408 g,0.6036 mmol, 54.4%). ESI-MS m/z calc. 398.1, found 399.0 (M+1)⁺.Retention time of 3.77 minutes. ¹H NMR (400 MHz, CD₃CN) d 1.21 (q, J=3.6Hz, 2H), 1.59 (q, J=3.6 Hz, 2H), 3.78 (s, 3H), 4.19 (s, 2H), 6.88-6.96(m, 2H), 7.07 (s, 1H), 7.23-7.46 (m, 6H).

Example 24

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-(2-methoxy-pyridin-3-ylmethyl)-thiazol-2-yl]-amide.5-(2-Methoxy-pyridin-3-ylmethyl)-thiazol-2-ylamine (221 mg, 1.00 mmol)and 1-benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (206 g, 1.00mmol) were dissolved in acetonitrile (5 mL) containing triethylamine(0.421 mL, 3.00 mmol).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 760 mg, 2.0 mmol) was added and the solutionwas allowed to stir at 78° C. for 12 hours. An aliquot of the crudeproduct was purified by reverse-phase preparative liquid chromatography.ESI-MS m/z calc. 409.1, found; 410.3 (M+1)⁺ Retention time 3.23 minutes.

Example 25

1-(3-Methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-methoxy-pyridin-3-ylmethyl)-thiazol-2-yl]-amide.5-(2-Methoxy-pyridin-3-ylmethyl)-thiazol-2-ylamine (221 mg, 1.00 mmol)and 1-(4-methoxy-phenyl)-cyclopropanecarboxylic acid (192 g, 1.00 mmol)were dissolved in acetonitrile (5 mL) containing triethylamine (0.421mL, 3.00 mmol). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 760 mg, 2.0 mmol) was added and the solutionwas allowed to stir at 78° C. for 12 hours. An aliquot of the crudeproduct was purified by reverse-phase preparative liquid chromatography.ESI-MS m/z calc. 395.1, found; 396.3 (M+1)⁺ Retention time 3.27 minutes.

Example 26

General Procedure: One equivalent of the appropriate carboxylic acid andone equivalent of the appropriate amine were dissolved in acetonitrilecontaining triethylamine (3 equivalents).Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP) was added and the solution was allowed to stir. The crude productwas purified by reverse-phase preparative liquid chromatography to yieldthe pure product.

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-(2-chloro-benzyl)-[1,3,4]thiadiazol-2-yl]-amide5-(2-Chloro-benzyl)-[1,3,4]thiadiazol-2-ylamine (23 mg, 0.10 mmol) and1-benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (21 mg, 0.10 mmol)were dissolved in acetonitrile (1.5 mL) containing triethylamine (42 □L,0.30 mmol). Benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP, 49 mg, 0.11 mmol) was added and the solutionwas allowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography to yield the pureproduct (5.7 mg, 0.014 mmol, 14%). ESI-MS m/z calc. 413.1, found; 414.3(M+1)⁺ Retention time 3.38 minutes.

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-(2-chloro-benzyl)-[1,3,4]oxadiazol-2-yl]-amide5-(2-Chloro-benzyl)-[1,3,4]oxadiazol-2-ylamine (21 mg, 0.10 mmol) and1-benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (21 mg, 0.10 mmol)were dissolved in acetonitrile (1.5 mL) containing triethylamine (42 □L,0.30 mmol). Benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP, 49 mg, 0.11 mmol) was added and the solutionwas allowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography to yield the pureproduct (6.0 mg, 0.015 mmol, 15%). ESI-MS m/z calc. 397.1, found; 398.3(M+1)⁺ Retention time 2.96 minutes.

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-(2-chloro-benzyl)-1-methyl-1H-imidazol-2-yl]-amide5-(2-Chloro-benzyl)-1-methyl-1H-imidazol-2-ylamine (21 mg, 0.10 mmol)and 1-benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (22 mg, 0.10mmol) were dissolved in acetonitrile (1.5 mL) containing triethylamine(42 □L, 0.30 mmol). Benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP, 49 mg, 0.11 mmol) was added and the solutionwas allowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography to yield the pureproduct as a trifluoroacetic acid salt (11 mg, 0.022 mmol, 22%). ESI-MSm/z calc. 409.1, found; 410.1 (M+1)⁺ Retention time 2.40 minutes.

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-(2-chloro-benzyl)-oxazol-2-yl]-amide5-(2-Chloro-benzyl)-oxazol-2-ylamine (21 mg, 0.10 mmol) and1-benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (22 mg, 0.10 mmol)were dissolved in acetonitrile (1.5 mL) containing triethylamine (42 □L,0.30 mmol). Benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP, 49 mg, 0.11 mmol) was added and the solutionwas allowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography to yield the pureproduct (15 mg, 0.037 mmol, 37%). ESI-MS m/z calc. 396.1, found; 396.6(M+1)⁺ Retention time 3.17 minutes.

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-(2-chloro-benzyl)-thiazol-2-yl]-amide.5-(2-Chloro-benzyl)-thiazol-2-ylamine (22 mg, 0.10 mmol) and1-benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (22 mg, 0.10 mmol)were dissolved in acetonitrile (1.5 mL) containing triethylamine (42 □L,0.30 mmol). Benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP, 49 mg, 0.11 mmol) was added and the solutionwas allowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography to yield the pureproduct (4.1 mg, 0.0098 mmol, 9.8%). ESI-MS m/z calc. 412.1, found;413.3 (M+1)⁺ Retention time 3.12 minutes.

1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid(4-phenyl-thiazol-2-yl)-amide1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid (2.18 g, 11.4 mmol) and4-phenyl-thiazol-2-ylamine (2.00 g, 11.4 mmol) were dissolved inacetonitrile (50 mL) containing triethylamine (3.17 mL, 22.8 mmol).Benzotriazol-1-yloxy tris(dimethylamino)-phosphonium hexafluorophosphate(BOP, 4.95 g, 11.4 mmol) was added and the solution was allowed to stirfor 64 hours. The reaction mixture was evaporated to dryness andpurified by column chromatography on silica gel using a gradient of5-20% ethyl acetate in hexanes. The pure fractions were combined andevaporated to dryness to yield a white solid (1.9 g, 5.43 mmol, 47.5%).ESI-MS m/z calc. 350.1, found 351.1 (M+1)⁺. Retention time of 3.68minutes. ¹H NMR (400 MHz, CD₃CN) δ 1.27 (q, J=3.6 Hz, 2H), 1.66 (q,J=3.6 Hz, 2H), 3.87 (s, 3H), 7.04 (m, 2H), 7.40 (m, 6H), 7.82 (m, 2H),8.79 (s, 1H).

Example 27

General Procedure: One equivalent of the appropriate carboxylic acid wasplaced in an oven-dried flask under nitrogen. A minimum of thionylchloride and a catalytic amount of and N,N-dimethylformamide was addedand the solution was allowed to stir for 30 minutes at room temperature.The excess thionyl chloride was removed under vacuum and the resultingsolid was suspended in a minimum of anhydrous 1,4-dioxane. This solutionwas slowly added to a stirred solution of one equivalent the appropriateaminoheterocycle dissolved in a minimum of anhydrous 1,4-dioxanecontaining three equivalents of triethylamine. The resulting mixture wasallowed to stir at room temperature for several hours. The mixture wasfiltered, evaporated to dryness, and then purified by columnchromatography.

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-(2-methoxy-benzyl)-thiazol-2-yl]-amide1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (1.87 g, 9.08 mmol)was dissolved in thionyl chloride (5 mL) under nitrogen for 30 minutes.A catalytic amount of N,N-dimethylformamide was added and stirringcontinued for an additional 30 minutes. The excess thionyl chloride wasevaporated and the resulting residue was dissolved in 1,4 dioxane (15mL). This solution was slowly added under nitrogen to5-(2-methoxy-benzyl)-thiazol-2-ylamine (2.00 g, 9.08 mmol) dissolved in1,4 dioxane (20 mL) containing triethylamine (3.5 mL, 25 mmol). Thesolution was allowed to stir for 2 hours. The reaction mixture wasfiltered, the precipitate was washed three times with 1,4 dioxane (20mL), and the combined filtrate was evaporated to dryness and purified bycolumn chromatography on silica gel using a gradient of 0-30% ethylacetate in hexanes. The pure fractions were combined and evaporated todryness to yield an off-white solid. The product was recrystallizedtwice from ethyl acetate/hexanes to yield the pure product (2.01 g, 4.92mmol, 54.2%). ESI-MS m/z calc. 408.11, found 409.3 (M+1)⁺. Retentiontime of 3.48 minutes. ¹H NMR (400 MHz, DMSO-d₆) δ 1.11 (m, 2H), 1.43 (m,2H), 3.80 (s, 3H), 3.97 (s, 2H), 6.01 (s, 2H), 6.87 (m, 3H), 6.98 (m,2H), 7.16 (m, 2H), 7.22 (m, 1H), 10.76 (s, 1H).

Example 28

General Procedure: One equivalent of the appropriate halide is mixedwith one equivalent of the appropriate nitrogen containing heterocyclicamide with one equivalent of sodium hydride in anhydrous tetrahydrofuran(THF). The reaction mixture was then subjected to microwave irradiationfor 10 minutes at 100° C. The solvent was evaporated to dryness and thecrude mixture was purified by reverse-phase preparative liquidchromatography to yield the pure product.

Specific Example

1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-chloro-benzyl)-3-(2-diethylamino-ethyl)-3H-thiazol-2-ylidene]-amideand 1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-chloro-benzyl)-thiazol-2-yl]-(2-diethylamino-ethyl)-amide1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-chloro-benzyl)-thiazol-2-yl]-amide (39.9 mg, 0.100 mmol) and(2-bromo-ethyl)-diethyl-amine hydrobromide (26.1 mg, 0.100 mmol) weredissolved in 1 mL of tetrahydrofuran. Sodium hydride (60% dispersion inoil, 8.8 mg, 0.22 mmol) was added and the reaction was subjected tomicrowave irradiation for 10 minutes at 100° C. The solvent wasevaporated to dryness and the crude mixture was purified byreverse-phase preparative liquid chromatography to yield1-(4-methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-chloro-benzyl)-3-(2-diethylamino-ethyl)-3H-thiazol-2-ylidene]-amide(10 mg, 0.020 mmol, 20%) ESI-MS m/z calc. 497.2, found 498.3 (M+1)⁺.Retention time of 2.64 minutes. ¹H NMR (400 MHz, CD₃CN) δ 1.07-1.17 (m,8H), 1.54-1.59 (m, 2H), 2.88-2.94 (m, 4H), 3.19-3.24 (m, 2H), 3.80 (s,3H), 4.07 (s, 2H), 4.24 (t, J=6.4 Hz, 2H), 6.86-6.91 (m, 2H), 6.97 (s,1H), 7.26-7.47 (m, 6H) and 1-(4-methoxy-phenyl)-cyclopropanecarboxylicacid [5-(2-chloro-benzyl)-thiazol-2-yl]-(2-diethylamino-ethyl)-amide (14mg, 0.028 mmol, 28%) ESI-MS m/z calc. 497.2, found 498.3 (M+1)⁺.Retention time of 2.65 minutes. ¹H NMR (400 MHz, CD₃CN) δ 1.08-1.17 (m,6H), 1.30-1.37 (m, 2H), 1.51-1.57 (m, 2H), 2.55-2.59 (m, 2H), 3.00-3.05(m, 4H), 3.79 (s, 3H), 4.26 (s, 2H), 4.42 (t, J=3.0 Hz, 2H), 6.90-6.95(m, 2H), 7.16-7.23 (m, 2H), 7.27-7.50 (m, 5H).

Example 29

General Procedure: The appropriate alcohol (one equivalent) was placedin a minimum of trifluoroacetic acid. One equivalent of the appropriatethiol was added and the reaction was allowed to stir for 16 hours. Themixture was then evaporated to dryness and purified by reverse-phasepreparative liquid chromatography to yield the pure product.

Specific Example

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid{5-[(2-chloro-phenyl)-(2-dimethylamino-ethylsulfanyl)-methyl]-thiazol-2-yl}-amide1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-[(2-chloro-phenyl)-hydroxy-methyl]-thiazol-2-yl]-amide (42 mg, 0.10mmol) was placed in 1 mL of trifluoroacetic acid.2-Dimethylamino-ethanethiol hydrochloride (14 mg, 0.10 mmol) was addedand the solution was allowed to stir for 16 hours at room temperature.The mixture was then evaporated to dryness and purified by reverse-phasepreparative liquid chromatography to yield the pure product as thetrifluoracetic acid salt (35 mg, 0.056 mol, 56%). ESI-MS m/z calc.515.1, found; 516.3 (M+1)⁺ Retention time 2.81 minutes. ¹H NMR (400 MHz,CD₃CN) δ 1.21-1.25 (m, 2H), 1.57-1.61 (m, 2H), 2.74 (s, 6H), 2.86 (t,J=7.9 Hz, 2H), 3.15-3.31 (m, 2H), 5.90 (s, 1H), 5.97 (s, 2H), 6.79 (d,J=7.9 Hz, 1H), 6.93-6.97 (m, 2H), 7.24 (s, 1H), 7.34 (t, J=6.8 Hz, 1H),7.41 (t, J=6.9 Hz, 1H), 7.46 (d, J=1.3 Hz, 1H), 7.73 (d, J=7.7 Hz, 1H).

Example 30

General Procedure: The appropriate alcohol (one equivalent) was placedin a minimum of anhydrous dichloromethane containing triethylamine (2equivalents). Methanesulfonyl chloride (one equivalent) was added andthe solution was stirred at room temperature for 1 hour. The appropriateamine (5 equivalents) was added and the solution was allowed to stir for16 hours at room temperature. The solution was then evaporated todryness and purified by reverse-phase preparative liquid chromatographyto yield the pure product.

Specific Example

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid{5-[(2-chloro-phenyl)-piperidin-1-yl-methyl]-thiazol-2-yl}-amide1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-[(2-chloro-phenyl)-hydroxy-methyl]-thiazol-2-yl]-amide (43 mg, 0.10mmol) was placed in 1 mL of anhydrous dichloromethane containingtriethylamine (28 □L, 0.20 mmol). Methanesulfonyl chloride (11 mg, 0.10mmol) was added and the solution was stirred at room temperature for 1hour. Piperidine (43 mg, 0.50 mmol) was added and the solution wasallowed to stir for 16 hours at room temperature. The solution was thenevaporated to dryness and purified by reverse-phase preparative liquidchromatography to yield the pure product as the trifluoracetic acid salt(11 mg, 0.018 mol, 18%). ESI-MS m/z calc. 495.1, found; 496.3 (M+1)⁺Retention time 2.52 minutes.

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid{5-[(2-chloro-phenyl)-(2-dimethylamino-ethylamino)-methyl]-thiazol-2-yl}-amide1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-[(2-chloro-phenyl)-hydroxy-methyl]-thiazol-2-yl]-amide (43 mg, 0.10mmol) was placed in 1 mL of anhydrous dichloromethane containingtriethylamine (28 □L, 0.20 mmol). Methanesulfonyl chloride (11 mg, 0.10mmol) was added and the solution was stirred at room temperature for 1hour. N,N-Dimethyl-ethane-1,2-diamine (44 mg, 0.50 mmol) was added andthe solution was allowed to stir for 16 hours at room temperature. Thesolution was then evaporated to dryness and purified by reverse-phasepreparative liquid chromatography to yield the pure product as thetrifluoracetic acid salt (20 mg, 0.040 mol, 40%). ESI-MS m/z calc.498.2, found; 499.3 (M+1)⁺ Retention time 2.43 minutes.

Example 31

General Procedure: The appropriate alcohol (one equivalent) was placedin a minimum of toluene containing p-toluenesulfonic acid (1.2equivalents). The appropriate alcohol (1.3 equivalents) was added andthe mixture was subjected to microwave irradiation for 5 minutes at 150°C. The mixture was then evaporated to dryness and purified byreverse-phase preparative liquid chromatography to yield the pureproduct.

Specific Example

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid{5-[(2-chloro-phenyl)-methoxy-methyl]-thiazol-2-yl}-amide1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid[5-[(2-chloro-phenyl)-hydroxy-methyl]-thiazol-2-yl]-amide (25 mg, 0.058mmol) was placed in 1 mL of toluene with p-toluenesulfonic acid (14 mg,0.073 mmol). Methanol (3.0 □L, 0.075 mmol) was added and the solutionwas subjected to microwave irradiation for 5 minutes at 150° C. Themixture was then evaporated to dryness and purified by reverse-phasepreparative liquid chromatography to yield the pure product. (6.0 mg,0.013 mol, 22%). ESI-MS m/z calc. 515.1, found; 443.3 (M+1)⁺ Retentiontime 3.72 minutes. ¹H NMR (400 MHz, CD₃CN) δ 1.23-1.30 (m, 2H),1.56-1.67 (m, 2H), 3.38 (s, 3H), 5.88 (s, 1H), 5.99 (s, 2H), 6.84 (d,J=6.2 Hz, 1H), 6.92-6.98 (m, 2H), 7.26 (s, 1H), 7.33-7.38 (m, 1H),7.41-7.48 (m, 2H), 7.62-7.71 (m, 1H).

Example 32

General Procedure: The appropriate aryl halide (1 equivalent) wasdissolved in a minimum of ethanol containing potassium hydroxide (2equivalents). The appropriate thiol (1 equivalent) was added and themixture is subjected to microwave irradiation for 15 minutes at 115° C.The crude reaction mixture was then separated by reverse-phasepreparative liquid chromatography to yield the pure product.

Specific Example

1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-chloro-phenylsulfanyl)-thiazol-2-yl]-amide1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid(5-bromo-thiazol-2-yl)-amide (35 mg, 0.10 mmol) was dissolved in ethanol(0.50 mL) containing potassium hydroxide (11 mg, 0.20 mmol).2-Chloro-benzenethiol (11 □L, 0.10 mmol) is added and the mixture issubjected to microwave irradiation for 15 minutes at 115° C. The crudereaction mixture is then separated by reverse-phase preparative liquidchromatography to yield the pure product (11 mg, 0.026 mmol, 26%).ESI-MS m/z calc. 416.0, found; 417.1 (M+1)⁺ Retention time 4.03 minutes.

Example 33

General Procedure: The appropriate sulfide (1 equivalent) was dissolvedin a minimum of acetic acid containing hydrogen peroxide (3equivalents). The mixture was warmed to 85° C. and carefully monitoredby liquid chromatography/mass spectrometry (LC/MS). When the startingmaterial was consumed ether and water was added to the mixture. Thelayers were separated, and the organic layer was dried over sodiumsulfate, filtered, and evaporated to dryness. The crude reaction mixturewas then separated by reverse-phase preparative liquid chromatography toyield the pure product.

Specific Examples

1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-chloro-benzenesulfinyl)-thiazol-2-yl]-amide1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-chloro-phenylsulfanyl)-thiazol-2-yl]-amide (50.0 mg, 0.120 mmol)was dissolved in 3 mL of acetic acid containing hydrogen peroxide (30%aqueous solution, 40.8 mg, 0.360 mmol). The mixture was warmed to 85° C.for 1 hour. Ether (1.5 mL) and water (1.5 mL) was added and the layerswere separated. the organic layer was dried over sodium sulfate,filtered, and evaporated to dryness. The crude reaction mixture was thenseparated by reverse-phase preparative liquid chromatography to yieldthe pure product (20.9 mg, 0.0483 mmol, 40.2%). ESI-MS m/z calc. 432.0,found; 433.3 (M+1)⁺ Retention time 3.34 minutes.

1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-chloro-benzenesulfinyl)-thiazol-2-yl]-amide1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid[5-(2-chloro-phenylsulfanyl)-thiazol-2-yl]-amide (50.0 mg, 0.120 mmol)was dissolved in 3 mL of acetic acid containing hydrogen peroxide (30%aqueous solution, 40.8 mg, 0.360 mmol). The mixture was warmed to 85° C.for 1 hour. Ether (1.5 mL) and water (1.5 mL) was added and the layerswere separated. the organic layer was dried over sodium sulfate,filtered, and evaporated to dryness. The crude reaction mixture was thenseparated by reverse-phase preparative liquid chromatography to yieldthe pure product (19.7 mg, 0.0439 mmol, 36.6%). ESI-MS m/z calc. 448.0,found; 449.1 (M+1)⁺ Retention time 3.53 minutes. ¹H NMR (400 MHz, CD₃CN)δ 1.23-1.35 (m, 2H), 1.61-1.68 (m, 2H), 3.80 (s, 3H), 6.90-7.00 (m, 2H),7.34-7.41 (m, 2H), 7.54-7.68 (m, 3H), 8.03-8.10 (m, 1H), 8.22-8.30 (m,1H), 9.32 (s, 1H).

Analytical data is provided for compounds of Table 1 in Table 2 below.TABLE 2 LC/MS LC/MS Cmpd # M⁺ RT (min) 1 322.10 2.16 2 350.00 2.58 3321.20 1.98 4 399.20 3.74 5 369.20 3.71 6 397.20 3.95 7 293.00 2.96 8427.20 3.97 9 441.10 4.12 10 325.00 3.48 11 383.00 3.20 12 399.00 3.8013 399.00 3.79 14 364.80 3.57 15 394.80 3.55 16 351.20 4.69 17 351.204.59 18 385.10 3.84 19 437.10 3.80 20 427.30 3.94 21 431.30 4.07 22411.10 3.77 23 415.30 3.94 24 411.10 3.75 25 415.30 3.92 26 423.10 3.6927 411.10 3.64 28 415.30 3.82 29 407.10 3.85 30 411.30 4.02 31 399.103.69 32 403.50 3.85 33 378.90 3.69 34 383.10 3.89 35 417.10 4.03 36417.30 4.10 37 417.30 4.11 38 413.30 3.76 42 390.30 3.82 43 413.00 3.8344 415.00 3.85 45 403.30 3.10 46 359.10 3.82 47 359.10 3.74 48 355.103.47 49 339.30 3.75 50 433.30 3.34 51 449.10 3.53 52 401.30 3.83 53443.30 2.90 54 429.30 3.02 55 413.30 3.12 56 439.30 2.71 57 425.30 2.7858 409.30 2.95 59 434.30 2.48 60 420.30 2.58 61 404.50 2.75 62 396.102.34 63 289.30 2.88 64 399.00 3.42 65 399.00 3.47 66 395.00 3.25 67390.00 3.00 68 390.00 3.05 69 395.00 70 413.30 3.59 71 471.30 3.60 72498.30 2.64 73 498.30 2.64 74 498.30 2.80 75 408.00 2.83 76 365.10 3.6277 385.10 3.70 78 381.30 3.37 79 365.10 3.32 80 399.10 3.50 81 379.303.53 82 399.10 3.63 83 395.30 3.30 84 302.90 2.58 85 365.10 3.32 86429.30 3.00 87 445.10 3.20 88 417.30 3.70 89 417.30 3.73 90 336.10 2.7491 332.30 2.57 92 415.30 3.72 93 411.30 3.43 94 398.30 3.17 95 398.302.38 96 427.30 3.05 97 412.10 3.12 98 427.30 3.45 99 412.30 2.36 100441.30 2.88 101 431.30 3.84 102 431.30 3.82 103 427.10 3.20 104 397.103.77 105 441.30 3.25 106 344.90 3.21 107 403.10 3.97 108 403.10 3.94 109390.90 3.93 110 433.10 4.09 111 394.90 3.84 112 425.10 3.80 113 395.103.77 114 399.10 4.04 115 409.10 3.77 116 433.10 4.25 117 434.10 3.87 118443.30 3.38 119 436.10 2.79 120 381.30 3.89 121 381.30 3.85 122 385.104.14 123 419.10 4.34 124 429.10 3.40 125 433.10 4.05 126 392.90 3.82 127425.10 3.60 128 419.10 4.19 129 394.90 3.64 130 517.30 3.44 131 517.303.34 132 546.30 2.57 133 517.10 3.66 134 503.30 3.27 135 427.30 3.46 136429.50 3.17 137 544.30 2.95 138 516.30 2.81 139 532.10 2.75 140 488.202.87 141 530.10 3.16 142 497.30 2.53 143 456.30 2.43 144 511.50 2.87 145503.10 3.37 146 489.10 3.29 147 375.10 2.87 148 361.10 2.90 149 417.303.77 150 349.10 3.00 151 417.30 3.75 152 383.30 3.20 153 400.30 3.40 154383.30 3.23 155 379.10 3.05 156 379.10 3.52 157 413.30 3.72 158 431.303.72 159 413.30 3.71 160 431.50 3.74 161 363.30 2.98 162 431.50 3.69 163396.90 3.17 164 431.30 3.69 165 397.30 3.18 166 414.30 3.38 167 397.103.20 168 392.90 3.03 169 410.10 2.40 170 398.30 2.96 171 449.00 3.80 172411.00 3.69 173 453.00 3.98 174 415.00 3.90 175 396.30 3.27 176 410.303.23 177 395.00 3.58 178 413.00 3.64 179 395.00 3.54 180 409.00 3.54 181427.00 3.60 182 409.00 3.48 183 389.30 3.94 184 496.30 2.52 185 510.502.62 186 499.30 2.43 187 541.50 2.41 188 382.30 2.47 189 357.30 2.86 190375.00 3.07 191 402.00 3.11 192 413.30 3.62 193 409.50 3.67 194 467.303.84 195 427.10 3.58 196 423.10 3.64 197 401.10 3.60 198 405.10 3.75 199409.30 3.47 200 413.30 3.63 201 481.00 3.78 202 498.30 2.92 203 418.102.63 204 430.10 3.30 205 445.30 2.28 206 487.50 2.29 207 366.70 2.71 208381.10 2.68 209 422.90 3.63 210 496.30 2.60 211 496.30 2.60 212 541.302.50 213 486.30 2.68 214 482.30 2.76 215 372.30 3.29 216 526.10 3.57 217482.30 3.00 218 526.30 3.03 219 516.30 2.71 220 526.30 2.88 221 470.302.85 222 526.30 2.83 223 512.30 2.78 224 539.30 3.22 225 526.10 3.07 226498.10 2.76 227 554.30 3.05 228 512.10 2.81 229 512.30 2.81 230 525.302.86 231 510.30 3.02 232 498.10 3.05 233 526.30 2.97 234 539.50 2.86 235555.30 2.61 236 485.30 2.76 237 555.30 2.81 238 537.50 2.90 239 457.303.86 240 478.90 2.55 241 395.90 2.21 242 420.90 3.69 243 410.30 2.19 244435.30 3.67 245 417.30 3.50 246 542.00 2.73 249 556.20 2.90 250 469.204.09 251 471.20 4.25 252 540.40 2.81 253 500.00 2.71 254 526.20 2.76 255526.20 3.30 256 526.20 3.08 257 513.10 4.00 258 513.30 4.09 259 443.303.72 260 471.30 4.02 261 400.30 3.02 262 396.30 2.40 263 401.30 3.63 264414.30 2.96 265 410.30 2.38 266 413.10 3.33 267 413.10 3.33 268 415.303.58 269 399.10 3.32 270 570.30 2.82 271 473.10 3.19 272 487.30 3.62 273487.30 3.27 274 501.30 3.34 275 514.10 2.66 276 556.30 2.54 277 570.102.61 278 584.30 2.67 279 379.10 3.72 280 393.10 3.65 281 397.30 3.74 282411.10 3.70 283 443.00 3.66 LC-MS LC-RT Cmpd # M+ min 284 364.11 2.65285 395.10 2.81 286 429.11 2.98 287 429.06 3.26 288 427.05 3.16 289344.08 2.72 290 386.13 3.12 291 437.17 2.69 292 303.09 2.37 293 365.082.73 294 378.10 2.92 295 375.16 2.66 296 380.12 3.06 297 375.16 2.49 298429.10 3.15 299 365.08 2.15 300 359.10 2.66 301 407.10 3.11 302 416.053.01 303 425.10 1.36 304 423.09 2.33 305 428.11 3.13 306 428.11 3.06 307435.09 2.33 308 461.06 2.37 309 446.05 2.37 310 498.30 2.33 311 498.302.35 312 468.14 1.40 313 453.13 2.75 314 466.16 1.46 315 453.13 2.76LC/MS LC/MS Cmpd # M⁺ RT (min) 316 384.07 2.44 317 384.07 2.42 318455.30 1.95 319 388.30 1.90 320 388.30 1.92

Example 34

B) Assays for Detecting and Measuring ΔF508-CFTR Correction Propertiesof Compounds

I) Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The optical membrane potential assay utilized voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission were monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with ΔF508-CFTR; a single-addition HTS assay format wasdeveloped. The cells were incubated in serum-free medium for 16 hrs at37° C. in the presence or absence (negative control) of test compound.As a positive control, cells plated in 384-well plates were incubatedfor 16 hrs at 27° C. to “temperature-correct” ΔF508-CFTR. The cells weresubsequently rinsed 3× with Krebs Ringers solution and loaded with thevoltage-sensitive dyes. To activate ΔF508-CFTR, 10 μM forskolin and theCFTR potentiator, genistein (20 μM), were added along with Cl⁻-freemedium to each well. The addition of Cl⁻-free medium promoted Cl⁻ effluxin response to ΔF508-CFTR activation and the resulting membranedepolarization was optically monitored using the FRET-basedvoltage-sensor dyes.

Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. During the first addition, a Cl⁻-free medium withor without test compound was added to each well. After 22 sec, a secondaddition of Cl⁻-free medium containing 2-10 μM forskolin was added toactivate ΔF508-CFTR. The extracellular Cl⁻ concentration following bothadditions was 28 mM, which promoted Cl⁻ efflux in response to ΔF508-CFTRactivation and the resulting membrane depolarization was opticallymonitored using the FRET-based voltage-sensor dyes.

Solutions

-   Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES    10, pH 7.4 with NaOH.-   Chloride-free bath solution: Chloride salts in Bath Solution #1 are    substituted with gluconate salts.-   CC2-DMPE: Prepared as a 10 mM stock solution in DMSO and stored at    −20° C.-   DiSBAC₂(3): Prepared as a 10 mM stock in DMSO and stored at −20° C.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at 30,000/well in 384-well matrigel-coatedplates and cultured for 2 hrs at 37° C. before culturing at 27° C. for24 hrs. for the potentiator assay. For the correction assays, the cellsare cultured at 27° C. or 37° C. with and without compounds for 16-24hoursB) Electrophysiological Assays for assaying ΔF508-CFTR modulationproperties of compounds

1. Ussing Chamber Assay

Ussing chamber experiments were performed on polarized epithelial cellsexpressing ΔF508-CFTR to further characterize the ΔF508-CFTR modulatorsidentified in the optical assays. FRT^(ΔF508-CFTR) epithelial cellsgrown on Costar Snapwell cell culture inserts were mounted in an Ussingchamber (Physiologic Instruments, Inc., San Diego, Calif.), and themonolayers were continuously short-circuited using a Voltage-clampSystem (Department of Bioengineering, University of Iowa, Iowa, and,Physiologic Instruments, Inc., San Diego, Calif.). Transepithelialresistance was measured by applying a 2-mV pulse. Under theseconditions, the FRT epithelia demonstrated resistances of 4 KΩ/cm² ormore. The solutions were maintained at 27° C. and bubbled with air. Theelectrode offset potential and fluid resistance were corrected using acell-free insert. Under these conditions, the current reflects the flowof Cl⁻ through ΔF508-CFTR expressed in the apical membrane. The I_(SC)was digitally acquired using an MP100A-CE interface and AcqKnowledgesoftware (ν3.2.6; BIOPAC Systems, Santa Barbara, Calif.).

Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate ΔF508-CFTR,forskolin (10 μM) and the PDE inhibitor, IBMX (100 μM), were appliedfollowed by the addition of the CFTR potentiator, genistein (50 μM).

As observed in other cell types, incubation at low temperatures of FRTcells stably expressing ΔF508-CFTR increases the functional density ofCFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with 10 μM of the test compound for24 hours at 37° C. and were subsequently washed 3× prior to recording.The cAMP- and genistein-mediated I_(SC) in compound-treated cells wasnormalized to the 27° C. and 37° C. controls and expressed as percentageactivity. Preincubation of the cells with the correction compoundsignificantly increased the cAMP- and genistein-mediated I_(SC) comparedto the 37° C. controls.

Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane and was permeabilized with nystatin (360μg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate(titrated to pH 7.4 with NaOH) to give a large Cl⁻ concentrationgradient across the epithelium. All experiments were performed 30 minafter nystatin permeabilization. Forskolin (10 μM) and all testcompounds were added to both sides of the cell culture inserts. Theefficacy of the putative ΔF508-CFTR potentiators was compared to that ofthe known potentiator, genistein.

Solutions

-   Basolateral solution (in mM): NaCl (135), CaCl₂ (1.2), MgCl₂ (1.2),    K₂HPO₄ (2.4), KHPO₄ (0.6),    N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (10),    and dextrose (10). The solution was titrated to pH 7.4 with NaOH.-   Apical solution (in mM): Same as basolateral solution with NaCl    replaced with Na Gluconate (135).

Cell Culture

Fisher rat epithelial (FRT) cells expressing ΔF508-CFTR(FRT^(ΔF508-CFTR)) were used for Ussing chamber experiments for theputative ΔF508-CFTR modulators identified from our optical assays. Thecells were cultured on Costar Snapwell cell culture inserts and culturedfor five days at 37° C. and 5% CO₂ in Coon's modified Ham's F-12 mediumsupplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100μg/ml streptomycin. Prior to use for characterizing the potentiatoractivity of compounds, the cells were incubated at 27° C. for 16-48 hrsto correct for the ΔF508-CFTR. To determine the activity of correctionscompounds, the cells were incubated at 27° C. or 37° C. with and withoutthe compounds for 24 hours.

2. Whole-Cell Recordings

The macroscopic ΔF508-CFTR current (I_(ΔF508)) in temperature- and testcompound-corrected NIH3T3 cells stably expressing ΔF508-CFTR weremonitored using the perforated-patch, whole-cell recording. Briefly,voltage-clamp recordings of I_(ΔF508) were performed at room temperatureusing an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,Foster City, Calif.). All recordings were acquired at a samplingfrequency of 10 kHz and low-pass filtered at 1 kHz. Pipettes had aresistance of 5-6 MΩ when filled with the intracellular solution. Underthese recording conditions, the calculated reversal potential for Cl⁻(E_(Cl)) at room temperature was −28 mV. All recordings had a sealresistance >20 GΩ and a series resistance <15 MΩ. Pulse generation, dataacquisition, and analysis were performed using a PC equipped with aDigidata 1320 A/D interface in conjunction with Clampex 8 (AxonInstruments Inc.). The bath contained <250 μl of saline and wascontinuously perifused at a rate of 2 ml/min using a gravity-drivenperfusion system.

Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional ΔF508-CFTR in the plasma membrane, we used theabove-described perforated-patch-recording techniques to measure thecurrent density following 24-hr treatment with the correction compounds.To fully activate ΔF508-CFTR, 10 μM forskolin and 20 μM genistein wereadded to the cells. Under our recording conditions, the current densityfollowing 24-hr incubation at 27° C. was higher than that observedfollowing 24-hr incubation at 37° C. These results are consistent withthe known effects of low-temperature incubation on the density ofΔF508-CFTR in the plasma membrane. To determine the effects ofcorrection compounds on CFTR current density, the cells were incubatedwith 10 μM of the test compound for 24 hours at 37° C. and the currentdensity was compared to the 27° C. and 37° C. controls (% activity).Prior to recording, the cells were washed 3× with extracellularrecording medium to remove any remaining test compound. Preincubationwith 10 μM of correction compounds significantly increased the cAMP- andgenistein-dependent current compared to the 37° C. controls.

Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in I_(ΔF508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

Solutions

-   Intracellular solution (in mM): Cs-aspartate (90), CsCl (50), MgCl₂    (1), HEPES (10), and 240 μg/ml amphotericin-B (pH adjusted to 7.35    with CsOH).-   Extracellular solution (in mM): N-methyl-D-glucamine (NMDG)-Cl    (150), MgCl₂ (2), CaCl₂ (2), HEPES (10) (pH adjusted to 7.35 with    HCl).

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1× pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

3. Single-Channel Recordings

The single-channel activities of temperature-corrected ΔF508-CFTR stablyexpressed in NIH3T3 cells and activities of potentiator compounds wereobserved using excised inside-out membrane patch. Briefly, voltage-clamprecordings of single-channel activity were performed at room temperaturewith an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). Allrecordings were acquired at a sampling frequency of 10 kHz and low-passfiltered at 400 Hz. Patch pipettes were fabricated from Corning KovarSealing #7052 glass (World Precision Instruments, Inc., Sarasota, Fla.)and had a resistance of 5-8 MΩ when filled with the extracellularsolution. The ΔF508-CFTR was activated after excision, by adding 1 mMMg-ATP, and 75 nM of the cAMP-dependent protein kinase, catalyticsubunit (PKA; Promega Corp. Madison, Wis.). After channel activitystabilized, the patch was perifused using a gravity-drivenmicroperfusion system. The inflow was placed adjacent to the patch,resulting in complete solution exchange within 1-2 sec. To maintainΔF508-CFTR activity during the rapid perifusion, the nonspecificphosphatase inhibitor F (10 mM NaF) was added to the bath solution.Under these recording conditions, channel activity remained constantthroughout the duration of the patch recording (up to 60 min). Currentsproduced by positive charge moving from the intra- to extracellularsolutions (anions moving in the opposite direction) are shown aspositive currents. The pipette potential (V_(p)) was maintained at 80mV.

Channel activity was analyzed from membrane patches containing ≦2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

Solutions

-   Extracellular solution (in mM): NMDG (150), aspartic acid (150),    CaCl₂ (5), MgCl₂ (2), and HEPES (10) (pH adjusted to 7.35 with Tris    base).-   Intracellular solution (in mM): NMDG-Cl (150), MgCl₂ (2), EGTA (5),    TES (10), and Tris base (14) (pH adjusted to 7.35 with HCl).

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, βP-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Compounds of the invention are useful as modulators of ATP bindingcassette transporters. Table 3 below illustrates the EC50 and relativeefficacy of exemplary embodiments of the present invention. Cmpd # EC50(uM) % Activity 1 ++ ++ 2 +++ +++ 3 +++ +++ 4 +++ +++ 5 +++ ++ 6 +++ +++7 +++ ++ 8 +++ +++ 9 +++ +++ 10 +++ +++ 11 ++ ++ 12 +++ +++ 13 +++ +++14 +++ +++ 15 +++ +++ 16 +++ +++ 17 +++ +++ 18 +++ +++ 19 +++ +++ 20 ++++++ 21 +++ +++ 22 +++ ++ 23 +++ +++ 24 +++ ++ 25 +++ +++ 26 +++ +++ 27+++ ++ 28 +++ +++ 29 +++ +++ 30 +++ +++ 31 +++ +++ 32 +++ +++ 33 +++ +++34 +++ +++ 35 +++ ++ 36 +++ ++ 37 +++ +++ 38 +++ +++ 42 +++ +++ 43 ++++++ 44 +++ +++ 45 +++ +++ 46 +++ +++ 47 +++ ++ 48 +++ +++ 49 +++ ++ 50+++ +++ 51 +++ ++ 52 +++ ++ 53 +++ ++ 54 +++ ++ 55 +++ +++ 56 +++ ++ 57+++ +++ 58 +++ +++ 59 +++ ++ 60 +++ ++ 61 +++ +++ 62 ++ ++ 63 +++ +++ 64+++ +++ 65 +++ +++ 66 +++ +++ 67 + ++ 68 +++ ++ 69 +++ +++ 70 +++ ++ 71++ ++ 72 + ++ 73 + + 74 + ++ 75 + ++ 76 +++ +++ 77 +++ +++ 78 +++ +++ 79+++ +++ 80 +++ +++ 81 +++ +++ 82 +++ +++ 83 +++ +++ 84 +++ +++ 85 ++++++ 86 +++ +++ 87 +++ +++ 88 +++ +++ 89 +++ +++ 90 +++ ++ 91 ++ ++ 92+++ ++ 93 +++ +++ 94 +++ +++ 95 +++ ++ 96 +++ ++ 97 +++ +++ 98 +++ +++99 +++ +++ 100 +++ ++ 101 +++ +++ 102 +++ +++ 103 + ++ 104 +++ +++ 105+++ +++ 106 +++ +++ 107 +++ +++ 108 +++ +++ 109 +++ +++ 110 +++ +++ 111+++ +++ 112 ++ + 113 +++ +++ 114 +++ +++ 115 +++ ++ 116 +++ +++ 117 ++++ 118 +++ ++ 119 +++ ++ 120 +++ +++ 121 +++ +++ 122 +++ +++ 123 +++ +++124 + ++ 125 +++ +++ 126 +++ +++ 127 +++ +++ 128 +++ +++ 129 +++ ++ 130+++ +++ 131 +++ ++ 132 +++ +++ 133 +++ +++ 134 +++ ++ 135 +++ ++ 136 ++++++ 137 +++ ++ 138 +++ ++ 139 +++ +++ 140 +++ ++ 141 +++ +++ 142 +++ ++143 +++ +++ 144 +++ +++ 145 +++ +++ 146 +++ +++ 147 +++ ++ 148 +++ ++149 +++ +++ 150 +++ ++ 151 +++ ++ 152 +++ ++ 153 +++ ++ 154 +++ ++ 155+++ ++ 156 +++ +++ 157 +++ +++ 158 +++ +++ 159 +++ +++ 160 +++ +++ 161+++ +++ 162 +++ +++ 163 +++ ++ 164 +++ +++ 165 +++ +++ 166 +++ +++ 167+++ ++ 168 +++ +++ 169 +++ ++ 170 +++ +++ 171 +++ +++ 172 +++ +++ 173+++ +++ 174 +++ +++ 175 +++ +++ 176 +++ +++ 177 +++ ++ 178 +++ +++ 179+++ +++ 180 +++ +++ 181 +++ +++ 182 +++ +++ 183 +++ +++ 184 +++ +++ 185+++ +++ 186 +++ ++ 187 +++ +++ 188 +++ ++ 189 +++ +++ 190 ++ + 191 +++++ 192 +++ +++ 193 +++ +++ 194 +++ ++ 195 +++ +++ 196 +++ +++ 197 ++++++ 198 +++ +++ 199 +++ +++ 200 +++ +++ 201 +++ ++ 202 +++ +++ 203 +++++ 204 +++ ++ 205 +++ ++ 206 +++ ++ 207 + ++ 208 +++ ++ 209 +++ +++ 210+++ +++ 211 +++ +++ 212 +++ +++ 213 +++ +++ 214 +++ +++ 215 +++ +++ 216+++ +++ 217 +++ +++ 218 +++ +++ 219 +++ +++ 220 +++ +++ 221 +++ +++ 222+++ +++ 223 +++ +++ 224 +++ +++ 225 +++ +++ 226 +++ +++ 227 +++ +++ 228+++ +++ 229 +++ +++ 230 +++ +++ 231 +++ +++ 232 +++ +++ 233 +++ +++ 234+++ +++ 235 +++ ++ 236 +++ +++ 237 +++ +++ 238 +++ +++ 239 +++ +++ 240+++ +++ 241 +++ ++ 242 +++ +++ 243 +++ +++ 244 +++ +++ 245 +++ +++ 246+++ +++ 249 +++ ++ 250 +++ +++ 251 +++ +++ 252 +++ ++ 253 +++ ++ 254 +++++ 255 +++ +++ 256 +++ +++ 257 +++ +++ 258 +++ +++ 259 +++ +++ 260 ++++++ 261 +++ +++ 262 +++ +++ 263 +++ +++ 264 +++ +++ 265 +++ +++ 266 ++++++ 267 +++ +++ 268 +++ +++ 269 +++ ++ 270 +++ ++ 271 +++ +++ 272 ++++++ 273 +++ +++ 274 +++ +++ 275 +++ + 276 +++ ++ 277 +++ ++ 278 +++ ++279 +++ +++ 280 +++ +++ 281 +++ +++ 282 +++ ++ 283 +++ ++In Table 3, the following meanings apply:EC50: “+++” means <10 uM; “++” means between 10 uM to 25 uM; “+” meansbetween 25 uM to 60 uM.% Efficacy: “+” means <25%; “++” means between 25% to 100%; “+++”means > 100%.

1. A compound having formula I:

or a pharmaceutically acceptable salt thereof, wherein: Ht is a 5-membered heteroaromatic ring containing 1-4 heteroatoms selected from O, S, N, or NH, wherein said ring is optionally fused to a 6-membered monocyclic or 10-membered bicyclic, carbocyclic or heterocyclic, aromatic or non-aromatic ring, wherein Ht is optionally substituted with w occurrences of —WR^(W), wherein w is 0-5; R^(N) is H or R; R is hydrogen or C₁₋₆ aliphatic wherein up to two methylene units of Q, W, or X are optionally and independently replaced by —CO—, —CS—, —COCO—, —CONR′—, —CONR′NR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—, —NR′NR′, —NR′NR′CO—, —NR′CO—, —S—, —SO, —SO₂—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—; ring A is 3-7 membered monocyclic ring having 0-3 heteroatoms selected from O, S, N, or NH, wherein ring A is optionally substituted with q occurrences of -QR^(Q); ring B is optionally fused to 5-6 membered carbocyclic or heterocyclic, aromatic or non-aromatic ring; each of x, q, and z is independently 0-5; each —X—R^(X), -Q-R^(Q), and -Z-R^(Z) is independently R′; R′ is independently R¹, R², R³, R⁴, or R⁵; R¹ is oxo, R⁶ or ((C1-C4)aliphatic)_(n)-Y; n is 0 or 1; Y is halo, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶, NH₂, NHR⁶, N(R⁶)₂, NR⁶R⁸, COOH, COOR⁶ or OR⁶; or two R¹ on adjacent ring atoms, taken together, form 1,2-methylenedioxy or 1,2-ethylenedioxy; R² is aliphatic, wherein each R² optionally comprises up to 2 substituents independently selected from R¹, R⁴, or R⁵; R³ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring optionally comprising up to 3 substituents, independently selected from R¹, R², R⁴ or R⁵; R⁴ is OR⁵, OR⁶, OC(O)R⁶, OC(O)R⁵, OC(O)OR⁶, OC(O)OR⁵, OC(O)N(R⁶)₂, OC(O)N(R⁵)₂, OC(O)N(R⁶R⁵), SR⁶, SR⁵, S(O)R⁶, S(O)R⁵, SO₂R⁶, SO₂R⁵, SO₂N(R⁶)₂, SO₂N(R⁵)₂, SO₂NR⁵R⁶, SO₃R⁶, SO₃R⁵, C(O)R⁵, C(O)OR⁵, C(O)R⁶, C(O)OR⁶, C(O)N(R⁶)₂, C(O)N(R⁵)₂, C(O)N(R⁵R⁶), C(O)N(OR⁶)R⁶, C(O)N(OR⁵)R⁶, C(O)N(OR⁶)R⁵, C(O)N(OR⁵)R⁵, C(NOR⁶)R⁶, C(NOR⁶)R⁵, C(NOR⁵)R⁶, C(NOR⁵)R⁵, N(R⁶)₂, N(R⁵)₂, N(R⁵R⁶), NR⁵C(O)R⁵, NR⁶C(O)R⁶, NR⁶C(O)R⁵, NR⁵C(O)R⁶, NR⁶C(O)OR⁶, NR⁵C(O)OR⁶, NR⁶C(O)OR⁵, NR⁵C(O)OR⁵, NR⁶C(O)N(R⁶)₂, NR⁶C(O)NR⁵R⁶, NR⁶C(O)N(R⁵)₂, NR⁵C(O)N(R⁶)₂, NR⁵C(O)NR⁵R⁶, NR⁵C(O)N(R⁵)₂, NR⁶SO₂R⁶, NR⁶SO₂R⁵, NR⁵SO₂R⁶,NR⁵SO₂R⁵, NR⁶SO₂N(R⁶)₂, NR⁶SO₂NR⁵R⁶, NR⁶SO₂N(R⁵)₂, NR⁵SO₂NR⁵R⁶, NR⁵SO₂N(R⁵)₂, N(OR⁶)R⁶, N(OR⁶)R⁵, N(OR⁵)R⁵, or N(OR⁵)R⁶; R⁵ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, optionally comprising up to 3 R¹ substituents; R⁶ is H or aliphatic, wherein R⁶ optionally comprises a R⁷ substituent; R⁷ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and each R7 optionally comprises up to 2 substituents independently chosen from H, (C1-C6)-straight or branched alkyl, (C₂-₆) straight or branched alkenyl or alkynyl, 1,2-methylenedioxy, 1,2-ethylenedioxy, or (CH₂)_(n)-Z; Z is selected from halo, CN, NO₂, CF₃, OCF₃, OH, S-aliphatic, S(O)-aliphatic, SO₂-aliphatic, NH₂, NH-aliphatic, N(aliphatic)₂, N(aliphatic)R⁸, NHR⁸, COOH, C(O)O(-aliphatic), or O-aliphatic; and R⁸ is an amino protecting group; provided that compounds set forth in FIG. 1 are excluded.
 2. The compound according to claim 1, wherein R′ is independently selected from hydrogen or an optionally substituted group selected from a C₁-C₈ aliphatic group, a 3-8-membered saturated, partially unsaturated, or fully unsaturated monocyclic ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-12 membered saturated, partially unsaturated, or fully unsaturated bicyclic ring system having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or two occurrences of R′ are taken together with the atom(s) to which they are bound to form an optionally substituted 3-12 membered saturated, partially unsaturated, or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
 3. The compound according to claim 1 or 2, wherein each of Q, X, and W is independently a bond or is an optionally substituted C₁-C₆ alkylidene chain wherein up to two methylene units of Q, W, or X are optionally and independently replaced by —CO—, —CS—, —COCO—, —CONR′—, —CONR′NR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—, —NR′NR′, —NR′NR′CO—, —NR′CO—, —S—, —SO, —SO₂—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—.
 4. The compound according to any one of claims 1-3, wherein each of R^(X), R^(Q), and R^(W) is independently R′, halo, NO₂, CN, CF₃, or OCF₃.
 5. The compound according to any one of claims 1-4, wherein Q is independently a bond or is an optionally substituted C₁₋₆ alkylidene chain wherein one or two non-adjacent methylene units are optionally and independently replaced by O, NR, S, SO₂, COO, or CO, and R^(Q) is R′ or halogen. In still other embodiments, each occurrence of QR^(Q) is independently —C₁₋₃alkyl, —O(C₁₋₃alkyl), —CF₃, —OCF₃, —SCF₃, —F, —Cl, —Br, or —COOR′, —COR′, —O(CH₂)₂N(R)(R′), —O(CH₂)N(R)(R′), —CON(R)(R′), —(CH₂)₂OR′, —(CH₂)OR′, optionally substituted phenyl, —N(R)(R′), —(CH₂)₂N(R)(R′), or —(CH₂)N(R)(R′).
 6. The compound according to any one of claims 1-4, wherein X is independently a bond or is an optionally substituted C₁₋₆ alkylidene chain wherein one or two non-adjacent methylene units are optionally and independently replaced by O, NR, S, SO₂, COO, or CO, and R^(X) is R′ or halogen. In still other embodiments, each occurrence of XR^(X) is independently —C₁₋₃alkyl, —O(C₁₋₃alkyl), —CF₃, —OCF₃, —SCF₃, —F, —Cl, —Br, or —COOR′, —COR′, —O(CH₂)₂N(R)(R′), —O(CH₂)N(R)(R′), —CON(R)(R′), —(CH₂)₂OR′, —(CH₂)OR′, optionally substituted phenyl, —N(R)(R′), —(CH₂)₂N(R)(R′), or —(CH₂)N(R)(R′).
 7. The compound according to any one of claims 1-4, wherein W is independently a bond or is an optionally substituted C₁₋₆ alkylidene chain wherein one or two non-adjacent methylene units are optionally and independently replaced by O, NR, S, SO₂, COO, or CO, and R^(W) is R′ or halogen. In still other embodiments, each occurrence of WR^(W) is independently —C₁₋₃alkyl, —O(C₁₋₃alkyl), —CF₃, —OCF₃, —SCF₃, —F, —Cl, —Br, or —COOR′, —COR′, —O(CH₂)₂N(R)(R′), —O(CH₂)N(R)(R′), —CON(R)(R′), —(CH₂)₂OR′, —(CH₂)OR′, optionally substituted phenyl, —N(R)(R′), —(CH₂)₂N(R)(R′), or —(CH₂)N(R)(R′).
 8. The compound according to any one of claims 1-7, wherein R is hydrogen.
 9. The compound according to any one of claims 1-8, wherein ring A is a 3-7 membered cycloalkyl ring.
 10. The compound according to any one of claims 1-8, wherein ring A is a 3-7 membered ring containing 1 heteroatom selected from O, NH, or S. Or, ring A contains up two heteroatoms selected from O, S, or NH.
 11. The compound according to claims 9 or 10, wherein ring A is selected from:


12. The compound according to any one of claims 1-11, wherein Ht is a 5-membered heteroaromatic ring containing 1-4 nitrogen atoms, wherein Ht is optionally substituted with up to three substituents.
 13. The compound according to claim 12, wherein said ring is optionally fused to a phenyl ring.
 14. The compound according to claim 12 or 13, wherein Ht is selected from one of the following rings:

wherein each ring is linked to the remainder of the molecule through a carbon ring atom.
 15. The compound according to any one of claims 1-14, wherein ring B is fused to a 5-6 membered carbocyclic or heterocyclic, aromatic or non-aromatic ring.
 16. The compound according to claim 15, wherein ring B is fused to a five membered heterocyclic ring.
 17. The compound according to claim 15, wherein ring B is fused to a five membered ring with 2 oxygen ring atoms.
 18. The compound according to any one of claims 1-14, wherein ring B is optionally substituted phenyl.
 19. The compound according to claim 1, having formula IIA:

wherein: m is 0-4; Ht₁ is a 5-membered heteroaromatic ring containing 1-4 heteroatoms selected from O, S, N, or NH, wherein said ring is optionally fused to a phenyl or 6-membered heteroaromatic ring; ring B is optionally fused to 5-6 membered carbocyclic or heterocyclic, aromatic or non-aromatic ring; X, R^(X), x, W, R^(W), and w are as defined in claim
 1. 20. The compound according to claim 19, wherein m is
 0. 21. The compound according to claim 19, wherein m is
 1. 22. The compound according to claim 19, wherein m is
 2. 23. The compound according to claim 19, wherein m is
 3. 24. The compound according to claim 19, wherein m is
 4. 25. The compound according to claim 19, wherein Ht¹ is selected from:

wherein each ring is linked to the remainder of the molecule through a carbon ring atom.
 26. The compound according to claim 25, wherein Ht₁ is selected from 1-d, 1-f, 1-j, 1-l, and 1-v rings above.
 27. The compound according to claim 26, wherein Ht₁ is 1-d.
 28. The compound according to claim 26, wherein Ht₁ is 1-f.
 29. The compound according to any one of claims 19-28, wherein w is
 1. 30. The compound according to any one of claims 19-29, wherein W—R^(W), taken together, forms —OR′ or R′ substituent.
 31. The compound according to any one of claims 19-30, wherein ring B is phenyl optionally substituted with up to x occurrences of X—R^(X).
 32. The compound according to any one of claims 19-31, wherein ring B is fused with a 5 memebered ring.
 33. The compound according to any one of claims 19-32, wherein x is 0-3.
 34. The compound according to any one of claims 19-32, wherein x is 0-2.
 35. The compound according to claim 1, having formula IIIA or formula IIIB:

wherein: m is 0 to 4; Ar is phenyl or a six-membered heteroaromatic ring; L is a bond, O, S, SO, SO₂, C(O), NR′, C₁₋₄ aliphatic, or CHR^(L); or L is

R^(L) is —OR′, —SR′, —SOR′, —SO₂R′, or —N(R′)₂; wherein ring A′ is a 3-7 membered monocyclic ring having 0-3 heteroatoms selected from O, S, N, or NH, wherein ring A′ is optionally substituted with q occurrences of -QR^(Q); R′ is as defined above; X₉ is CH₂ or CF₂; Ht₁ is a 5-membered heteroaromatic ring containing 1-4 heteroatoms selected from O, S, N, or NH, wherein said ring is optionally fused to a phenyl ring.
 36. The compound according to claim 35, wherein Ht¹ is selected from:

wherein each ring is linked to the remainder of the molecule through a carbon ring atom.
 37. The compound according to claim 35, wherein Ht₁ is 1-d, 1-f, 1-j, 1-l, and 1-v.
 38. The compound according to claim 35, wherein ring Ar is selected from:


39. The compound according to claim 35, wehrein m is
 0. 40. The compound according to claim 35, wherein m is
 1. 41. The compound according to claim 35, m is
 2. 42. The compound according to claim 35, m is
 3. 43. The compound according to claim 35, m is
 4. 44. The compound according to claim 35, wherein said compound has formula IVA′ or formula IVB′:

wherein: L is a bond, O, S, SO, SO₂, C(O), NR′, C₁₋₄ aliphatic, or CHR^(L); or L is

R^(L) is —OR′, —SR′, —SOR′, —SO₂R′, or —N(R′)₂; wherein ring A′ is a 3-7 membered monocyclic ring having 0-3 heteroatoms selected from O, S, N, or NH, wherein ring A′ is optionally substituted with q occurrences of -QR^(Q); R′ is as defined above; X₉ is CH₂ or CF₂; X¹ is O, S, or NR; R is hydrogen or C₁₋₄ aliphatic; and each of X² and X³ is independently selected from CH or N.
 45. The compound according to claim 44, having formula IVA-1′ or formula IVD-1′:


46. The compound according to claim 45, wherein X¹ is S, X² is N, and X³ is CH.
 46. The compound according to claim 45, wherein X¹ is S, X² and X³ both are N.
 47. The compound according to claim 45, wherein X¹ is S, X² is CH, and X³ is N.
 48. The compound according to claim 45, wherein X¹ is O, X² is N, and X³ is CH.
 49. The compound according to claim 45, wherein X¹ is O, X² and X³ both are N.
 50. The compound according to claim 45, wherein X¹ is O, X² is CH, and X³ is N.
 51. The compound according to claim 45, wherein X¹ is NR, X² is N, and X³ is CH.
 52. The compound according to claim 45, wherein R is hydrogen.
 53. The compound according to claim 45, wherein R is C₁₋₄ alkyl.
 54. The compound according to claim 45, wherein X¹ is NR, X² and X³ both are N, wherein R is hydrogen or R is C₁₋₄ alkyl.
 55. The compound according to claim 45, wherein X¹ is NR, X² is CH, and X³ is N, wherein R is hydrogen or C₁₋₄ alkyl.
 56. The compound according to claim 44, having formula IVA-2′ or formula IVB-1′:


57. The compound according to claim 56, wherein X¹ is S, X² is N, and X³ is CH.
 58. The compound according to claim 56, wherein X¹ is S, X² and X³ both are N.
 59. The compound according to claim 56, wherein X¹ is S, X² is CH, and X³ is N.
 60. The compound according to claim 56, wherein X¹ is O, X² is N, and X³ is CH.
 61. The compound according to claim 56, wherein X¹ is O, X² and X³ both are N.
 62. The compound according to claim 56, wherein X¹ is O, X² is CH, and X³ is N.
 63. The compound according to claim 56, wherein X¹ is NR, X² is N, and X³ is CH.
 64. The compound according to claim 56, wherein X¹ is NR, X² and X³ both are N.
 65. The compound according to claim 56, wherein X¹ is NR, X² is CH, and X³ is N.
 66. The compound according to claim 44, having formula IVA-3′ or formula IVB-3′:


67. The compound according to claim 66, wherein X¹ is S, X² is N, and X³ is CH.
 68. The compound according to claim 66, wherein X¹ is S, X² and X³ both are N.
 69. The compound according to claim 66, wherein X¹ is S, X² is CH, and X³ is N.
 70. The compound according to claim 66, wherein X¹ is O, X² is N, and X³ is CH.
 71. The compound according to claim 66, wherein X¹ is O, X² and X³ both are N.
 72. The compound according to claim 66, wherein X¹ is O, X² is CH, and X³ is N.
 73. The compound according to claim 66, wherein X¹ is NR, X² is N, and X³ is CH.
 74. The compound according to claim 66, wherein X¹ is NR, X² and X³ both are N.
 75. The compound according to claim 66, wherein X¹ is NR, X² is CH, and X³ is N.
 76. The compound according to claim 44, having formula IVA-4′ or formula IVD-4′:


77. The compound according to claim 76, wherein X¹ is S, X² is N, and X³ is CH.
 78. The compound according to claim 76, wherein X¹ is S, X² and X³ both are N.
 79. The compound according to claim 76, wherein X¹ is S, X² is CH, and X³ is N.
 80. The compound according to claim 76, wherein X¹ is O, X² is N, and X³ is CH.
 81. The compound according to claim 76, wherein X¹ is O, X² and X³ both are N.
 82. The compound according to claim 76, wherein X¹ is O, X² is CH, and X³ is N.
 83. The compound according to claim 76, wherein X¹ is NR, X² is N, and X³ is CH.
 84. The compound according to claim 76, wherein X¹ is NR, X² and X³ both are N.
 85. The compound according to claim 76, wherein X¹ is NR, X² is CH, and X³ is N.
 86. The compound according to claim 44, having formula IVA-5′ or formula IVA-6′:


87. The compound according to claim 86, wherein X¹ is S, X² is N, and X³ is CH.
 88. The compound according to claim 86, wherein X¹ is S, X² and X³ both are N.
 89. The compound according to claim 86, wherein X¹ is S, X² is CH, and X³ is N.
 90. The compound according to claim 86, wherein X¹ is O, X² is N, and X³ is CH.
 91. The compound according to claim 86, wherein X¹ is O, X² and X³ both are N.
 92. The compound according to claim 86, wherein X¹ is O, X² is CH, and X³ is N.
 93. The compound according to claim 86, wherein X¹ is NR, X² is N, and X³ is CH.
 94. The compound according to claim 86, wherein X¹ is NR, X² and X³ both are N.
 95. The compound according to claim 86, wherein X¹ is NR, X² is CH, and X³ is N.
 96. The compound according to claim 86, wherein X¹ is S, X² is N, and X³ is CH.
 97. The compound according to claim 86, wherein X¹ is O, X² is N, and X³ is CH.
 98. The compound according to claim 86, wherein X¹ is O, X² is CH, and X³ is N.
 99. The compound according to claim 44, having formula VA-1′, formula VA-2′, formula VA-3′, or formula VA-4′:

wherein: each of R^(C), R^(D), and R^(E) is independently hydrogen or C₁₋₆ aliphatic optionally substituted with —OH, —O(C₁₋₄aliphatic), —S(C₁₋₄aliphatic), —CF₃, —OCF₃, —SCF₃, halo, NH₂, NHR, N(R)₂, C(O)OH, C(O)O(C₁₋₄aliphatic), NHC(O)(C₁₋₄aliphatic), or a 3-7 membered heterocyclic ring containing up to 4 heteroatoms selected from O, N, or S, wherein said ring is optionally substituted with up to 2 substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y; p is 0 or 1; Y is halo, CN, NO₂, CF₃, OCF₃, OR, SR, NH₂, NHR, N(R)₂, COOH, or COOR; R is hydrogen or C₁₋₄ aliphatic; R^(AA) and R^(BB), taken together with the nitrogen atom, is a 3-7 membered heterocyclic ring containing up to 4 heteroatoms selected from O, N, or S, wherein said ring is optionally substituted with up to 2 substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y; p is 0 or 1; Y is halo, CN, NO₂, CF₃, OCF₃, OR, SR, NH₂, NHR, N(R)₂, COOH, or COOR; and R is hydrogen or C₁₋₄ aliphatic.
 100. The compound according to claim 99, wherein R^(C) is C₁₋₄ alkylidene optionally substituted with up to two substituents selected from —OH, —O(C₁₋₄alkyl), NH(C₁₋₄ alkyl), or N(C₁₋₄ alkyl)₂.
 101. The compound according to claim 99, wherein R^(C) is C₁₋₄ alkylidene optionally substituted with 5-6 membered heterocyclic ring containing up to 2 heteroatoms selected from O, N, or S, wherein said ring is optionally substituted with up to 2 substituents selected from oxo, (C₁₋₄ aliphatic), (C₁₋₄ aliphatic)-Y, wherein Y is halo, —OH, or —O(C₁₋₄ alkyl).
 102. The compound according to claim 99, wherein R^(C) is selected from —(CH₂)₂-(4-hydroxy-1-piperidyl), —(CH₂)₃-(4-hydroxy-1-piperidyl), —(CH₂)₄-(4-hydroxy-1-piperidyl), ethylmethylamino-ethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, 2-methoxyethyl, isopropyl, (2-methoxymethyl-1-pyrrolidinyl)ethyl, tetrahydrofuran-2-ylmethyl, (pyrrolidin-2-one-5-yl)methyl, (pyrrolidin-1-yl)ethyl, dimethylaminoethyl, 1-piperidylethyl, allyl, n-propyl, diisopropylaminoethyl, and (N-morpholino)ethyl.
 103. The compound of formula VA-1 according claim 99, wherein: a. z is 0-2 and ZR^(Z) together is selected from halo, OMe, OEt, CN, C(O)NH₂, Me, Et, NH₂, COOH, SO₂Me, N(Me)₂, or N(Et)₂; b. x is 0-2 and XR^(X) together is selected from halo, OMe, OEt, CN, C(O)NH₂, Me, Et, NH₂, COOH, SO₂Me, N(Me)₂, N(Et)₂, OCF₃, or SMe; and c. R^(C) is —(CH₂)₂-(4-hydroxy-1-piperidyl), —(CH₂)₃-(4-hydroxy-1-piperidyl), —(CH₂)₄-(4-hydroxy-1-piperidyl), (ethylmethylamino)ethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, 2-methoxyethyl, isopropyl, (2-methoxymethyl-1-pyrrolidinyl)ethyl, tetrahydrofuran-2-ylmethyl, (pyrrolidin-2-one-5-yl)methyl, (pyrrolidin-1-yl)ethyl, dimethylaminoethyl, 1-piperidylethyl, allyl, n-propyl, diisopropylaminoethyl, or (N-morpholino)ethyl.
 104. The compound according to claim 99, wherein R^(D) is C₁₋₄ alkylidene optionally substituted with —OH, —O(C₁₋₄alkyl), NH(C₁₋₄ alkyl), or N(C₁₋₄ alkyl)₂.
 105. The compound according to claim 99, wherein R^(D) is C₁₋₄ alkylidene optionally substituted with 5-6 membered heterocyclic ring containing up to 2 heteroatoms selected from O, N, or S, wherein said ring is optionally substituted with up to 2 substituents selected from oxo, (C₁₋₄ aliphatic), (C₁₋₄ aliphatic)-Y, wherein Y is halo, —OH, or —O(C₁₋₄ alkyl).
 106. The compound according to claim 99, wherein R^(D) is selected from (N-morpholino)ethyl, (N-morpholino)propyl, dimethylaminoethyl, (N-piperidyl)ethyl, allyl, or diisopropylaminoethyl.
 107. The compound of formula VA-2 according to claim 99, wherein: a. z is 0-2 and ZR^(Z) together is selected from halo, OMe, OEt, CN, C(O)NH₂, Me, Et, NH₂, COOH, SO₂Me, N(Me)₂, or N(Et)₂; b. x is 0-2 and XR^(X) together is selected from halo, OMe, OEt, CN, C(O)NH₂, Me, Et, NH₂, COOH, SO₂Me, N(Me)₂, N(Et)₂, OCF₃, or SMe; and c. R^(D) is (N-morpholino)ethyl, (N-morpholino)propyl, dimethylaminoethyl, (N-piperidyl)ethyl, allyl, or diisopropylaminoethyl
 108. The compound of formula VA-3 according to claim 99, wherein each of R^(AA) and R^(BB) is independently C₁₋₄ alkylidene optionally substituted with up to two substituents selected from —OH, —O(C₁₋₄alkyl), NH(C₁₋₄ alkyl), or N(C₁₋₄ alkyl)₂.
 109. The compound according to claim 108, wherein R^(AA) and R^(BB) is C₁₋₄ alkylidene optionally substituted with 5-6 membered heterocyclic ring containing up to 2 heteroatoms selected from O, N, or S, wherein said ring is optionally substituted with up to 2 substituents selected from oxo, (C₁₋₄ aliphatic), (C₁₋₄ aliphatic)-Y, wherein Y is halo, —OH, or —O(C₁₋₄ alkyl).
 110. The compound according to claim 109, wherein R^(AA) and R^(BB) is selected from Me, Et, propyl, butyl, allyl, hydroxyethyl, or dihydroxypropyl.
 111. The compound according to claim 108, wherein R^(AA) and R^(BB), taken together with the nitrogen atom, is selected from:


112. The compound according to claim 99, wherein R^(AB) is selected from methoxymethyl, methoxyethyl, allyl, methyl, —OH, hydroxymethyl, hydroxyethyl, ethylenedioxy, COOH, or C(O)CH₃.
 113. The compound of formula VA-4 according to claim 99, wherein R^(E) is independently selected from hydrogen or C₁₋₆ aliphatic optionally substituted with —OH, —O(C₁₋₄aliphatic), —S(C₁₋₄aliphatic), —CF₃, —OCF₃, —SCF₃, halo, NH₂, NHR, N(R)₂, C(O)OH, C(O)O(C₁₋₄aliphatic), or NHC(O)(C₁₋₄aliphatic).
 114. The compound according to claim 113, wherein R^(E) is selected from —(CH₂)₂OH, —(CH₂)₃OH, —(CH₂)₄OH, —(CH₂)₂NH₂, —(CH₂)₂NMe₂, —(CH₂)₂NEt₂, —(CH₂)₂NHC(O)Me, —(CH₂)COOH, —(CH₂)₂COOH, —(CH₂)COOMe, —(CH₂)CH(NH₂)COOH, or —(CH₂)₂CH(NH₂)COOH.
 115. The compound according to claim 44, having formula VC-1′, formula VC-2′, formula VC-3′, of formula VC-4′:

wherein: each of R^(C) and R^(D) is independently hydrogen or C₁₋₆ aliphatic optionally substituted with —O(C₁₋₄aliphatic), —CF₃, —OCF₃, —SCF₃, halo, NH₂, NHR, N(R)₂, or a 3-7 membered heterocyclic ring containing up to 4 heteroatoms selected from O, N, or S, wherein said ring is optionally substituted with up to 2 substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y; p is 0 or 1; Y is halo, CN, NO₂, CF₃, OCF₃, OR, SR, NH₂, NHR, N(R)₂, COOH, or COOR; R is hydrogen or C₁₋₄ aliphatic; R^(AA) and R^(BB), taken together with the nitrogen atom, is a 3-7 membered heterocyclic ring containing up to 4 heteroatoms selected from O, N, or S, wherein said ring is optionally substituted with up to 2 substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y; p is 0 or 1; Y is halo, CN, NO₂, CF₃, OCF₃, OR, SR, NH₂, NHR, N(R)₂, COOH, or COOR; and R is hydrogen or C₁₋₄ aliphatic.
 116. The compound according to claim 44, having formula VIA or formula VIB:

wherein: X¹ is O, S, or NR; and R is hydrogen or C₁₋₄ alkyl.
 117. The compound according to claim 116, wherein X¹ is S.
 118. The compound according to claim 116, wherein X¹ is O.
 119. The compound according to claim 116, wherein X¹ is NR.
 120. The compound of formula VIB according to claim 116, wherein X¹ is S.
 121. The compound of formula VIB according to claim 116, X¹ is O.
 122. The compound of formula VIB according to claim 116, X¹ is NR.
 123. The compound according to claim 44, having formula VIIA or formula VIIB:

wherein: m is 0-4; X⁸ is O or S; and R″ is C₁₋₄ alkyl.
 124. The compound according to claim 123, wherein m is
 0. 125. The compound according to claim 123, wherein, m is
 2. 126. The compound according to claim 123, wherein m is
 3. 127. The compound according to claim 123, wherein R″ is methyl.
 128. The compound according claim 123, wherein X⁸ is S. Or, X⁸ is O.
 129. A pharmaceutical composition comprising: (i) a compound according to any one of claims 1-128; and (ii) a pharmaceutically acceptable carrier.
 130. The composition of claim 129, optionally further comprising an additional agent selected from a mucolytic agent, bronchodialator, an anti-biotic, an anti-infective agent, an anti-inflammatory agent, CFTR corrector, or a nutritional agent.
 131. A method of increasing the number of functional ABC transporters in a membrane of a cell, comprising the step of contacting said cell with a compound according to any one of claims 1-128.
 132. The method of claim 131, wherein the ABC transporter is CFTR.
 133. A kit for use in measuring the activity of a ABC transporter or a fragment thereof in a biological sample in vitro or in vivo, comprising: (i) a first composition comprising a compound according to any one of claims 1-128; and (ii) instructions for: a) contacting the composition with the biological sample; b) measuring activity of said ABC transporter or a fragment thereof.
 134. The kit according to claim 133, further comprising instructions for a) contacting an additional composition with the biological sample; b) measuring the activity of said ABC transporter or a fragment thereof in the presence of said additional compound, and c) comparing the activity of the ABC transporter in the presence of the additional compound with the density of the ABC transporter in the presence of said first composition.
 135. The kit of claim 133, wherein the kit is used to measure the density of CFTR.
 136. A method of treating a condition, disease, or disorder in a patient implicated by ABC transporter activity, comprising the step of administering to said patient a compound according to any one of claims 1-128.
 137. The method according to claim 136, wherein said condition, disease, or disorder is selected from Cystic fibrosis, Hereditary emphysema, Hereditary hemochromatosis, Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency, Type 1 hereditary angioedema, Lipid processing deficiencies, such as Familial hypercholesterolemia, Type 1 chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, such as I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II, Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism, Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma, Glycanosis CDG type 1, Hereditary emphysema, Congenital hyperthyroidism, Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Progressive supranuclear plasy, Pick's disease, several polyglutamine neurological disorders asuch as Huntington, Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy, Dentatorubal pallidoluysian, and Myotonic dystrophy, as well as Spongiform encephalopathies, such as Hereditary Creutzfeldt-Jakob disease (due to Prion protein processing defect), Fabry disease, Straussler-Scheinker disease, secretory diarrhea, polycystic kidney disease, chronic obstructive pulmonary disease (COPD), dry eye disease, or Sjögren's Syndrome. 